Influenza hemagglutinin and neuraminidase variants

ABSTRACT

Polypeptides, polynucleotides, methods, compositions, and vaccines comprising (avian pandemic) influenza hemagglutinin and neuraminidase variants are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of application Ser. No.11/133,346 filed May 20, 2005, allowed, which claims the benefit under35 U.S.C. §119 (e) of U.S. Provisional Application Nos. 60/574,553 filedMay 25, 2004 and 60/657,554, filed Feb. 28, 2005, the disclosures ofeach of which are incorporated herein in their entirety for allpurposes.

BACKGROUND OF THE INVENTION

Vaccines against various and evolving strains of influenza are importantfrom a community health stand point, as well as commercially, since eachyear numerous individuals are infected with different strains and typesof influenza virus. Infants, the elderly, those without adequate healthcare and immuno-compromised persons are at special risk of death fromsuch infections. Compounding the problem of influenza infections is thatnovel influenza strains evolve readily and can spread amongst variousspecies, thereby necessitating the continuous production of newvaccines.

Numerous vaccines capable of producing a protective immune responsespecific for such different and influenza viruses/virus strains havebeen produced for over 50 years and include whole virus vaccines, splitvirus vaccines, surface antigen vaccines and live attenuated virusvaccines. However, while appropriate formulations of any of thesevaccine types are capable of producing a systemic immune response, liveattenuated virus vaccines have the advantage of also being able tostimulate local mucosal immunity in the respiratory tract. Considerablework in the production of influenza viruses, and fragments thereof, forproduction of vaccines has been done by the present inventors andco-workers; see, e.g., U.S. Application Nos. 60/420,708, filed Oct. 23,2002; 60/574,117, filed May 24, 2004; 10/423,828, filed Apr. 25, 2003;60/578,962, filed Jun. 12, 2004; and 10/870,690 filed Jun. 16, 2004, thedisclosure of which is incorporated by reference herein.

Because of the continual emergence (or re-emergence) of differentinfluenza strains, new influenza vaccines are continually desired. Suchvaccines typically are created using antigenic moieties of the newlyemergent virus strains, thus, polypeptides and polynucleotides of novel,newly emergent, or newly re-emergent virus strains (especially sequencesof antigenic genes) are highly desirable.

The present invention provides new and/or newly isolated influenzahemagglutinin and neuraminidase variants that are capable of use inproduction of numerous types of vaccines as well as in research,diagnostics, etc. Numerous other benefits will become apparent uponreview of the following.

SUMMARY OF THE INVENTION

In some aspects herein, the invention comprises an isolated orrecombinant polypeptide that is selected from: the polypeptides encodedby any one of the sequences of SEQ ID NO:1 through SEQ ID NO:10, any oneof the polypeptides encoded by SEQ ID NO:1 through SEQ ID NO:10; any oneof the polypeptides of SEQ ID NO:11 through SEQ ID NO:20; only the openreading frame of the polypeptides of SEQ ID NO:11 through SEQ ID NO:20;alternative (e.g., the mature form without the signal peptide, orwithout the 5′ and 3′ sequences outside of the open reading frame, orthe sequences as expressed on the surface of a virus (e.g., influenza))forms of the polypeptide of SEQ ID NO:11-20; any polypeptide that isencoded by a polynucleotide sequence which hybridizes under highlystringent conditions over substantially the entire length of apolynucleotide sequence of SEQ ID NO:1 through SEQ ID NO:10; anypolypeptide that is encoded by a polynucleotide sequence whichhybridizes under highly stringent conditions to a polynucleotidesequence of SEQ ID NO:1 through SEQ ID NO:10; and, a fragment of any ofthe above wherein the sequence comprises a hemagglutinin orneuraminidase polypeptide, or a fragment of a hemagglutinin orneuraminidase polypeptide, preferably where the fragments generate anantibody that specifically binds a full length polypeptide of theinvention. In various embodiments, the isolated or recombinantpolypeptides of the invention are substantially identical to about 300contiguous amino acid residues of any of the above polypeptides. In yetother embodiments, the invention comprises isolated or recombinantpolypeptides, that comprise an amino acid sequence that is substantiallyidentical over at least about 350 amino acids; over at least about 400amino acids; over at least about 450 amino acids; over at least about500 amino acids; over at least about 502 amino acids; over at leastabout 550 amino acids; over at least about 559 amino acids; over atleast about 565 amino acids; or over at least about 566 amino acidscontiguous of any of the above polypeptides. In some embodiments, thepolypeptide sequence (e.g., as listed in the sequence listing herein)comprises less than 565, 559, etc. amino acids. In such embodiments, theshorter listed polypeptides optionally comprise less than 565, 559, etc.amino acids. In yet other embodiments, the polypeptides of the inventionoptionally comprise fusion proteins, proteins with a leader sequence, aprecursor polypeptide, proteins with a secretion signal or alocalization signal, or proteins with an epitope tag, an E-tag, or a Hisepitope tag. In still other embodiments, the invention comprises apolypeptide comprising a sequence having at least 85%, at least 90%, atleast 93%, at least 95%, at least 98%, at least 98.5%, at least 99%, atleast 99.2%, at least 99.4%, at least 99.6%, at least 99.8%, or at least99.9% sequence identity to at least one polypeptide listed above. Thesequences of the invention are also shown in Appendix 1 and in thesequence listings herein. The hemagglutinin sequences of the inventioncan comprise both those sequences with unmodified and modified polybasiccleavage sites (thereby allowing growth of the viruses in eggs). Thehemagglutinin polypeptide sequences of SEQ ID NOS:11-20 comprise theendogenous amino terminal signal peptide sequences, however, thehemagglutinin polypeptide sequences of the invention also include themature (amino terminal signal peptide cleaved) form of the hemagglutininpolypeptides. The cleavage sites of any hemagglutinin polypeptidesequence of any influenza strain can be routinely measured or predictedusing any number of methods in the art.

In other aspects, the invention comprises a composition with one or morepolypeptide listed above, or fragments thereof. The invention alsoincludes polypeptides that are specifically bound by a polyclonalantisera raised against at least 1 antigen that comprises at least oneamino acid sequence described above, or a fragment thereof. Suchantibodies specific for the polypeptides described above are alsofeatures of the invention. The polypeptides of the invention areoptionally immunogenic.

The invention also encompasses immunogenic compositions comprising animmunologically effective amount of one or more of any of thepolypeptides described above as well as methods for stimulating theimmune system of an individual to produce a protective immune responseagainst influenza virus by administering to the individual animmunologically effective amount of any of the above polypeptides in aphysiologically acceptable carrier.

Additionally, the invention includes recombinant influenza virus thatcomprises one or more of the polypeptides or polynucleotides above, inaddition to immunogenic compositions comprising an immunologicallyeffective amount of such recombinant influenza virus. Methods forstimulating the immune system of an individual to produce a protectiveimmune response against influenza virus, through administering animmunologically effective amount of such recombinant influenza virus ina physiologically acceptable carrier are also part of the invention.

In other aspects, the invention comprises an isolated or recombinantnucleic acid that is selected from: any one of the polynucleotidesequences SEQ ID NO:1 through SEQ ID NO:10 (or complementary sequencesthereof), any one of the polynucleotide sequences encoding a polypeptideof SEQ ID NO:11 through SEQ ID NO:20 (or complementary polynucleotidesequences thereof), a polynucleotide sequence which hybridizes underhighly stringent conditions over substantially the entire length of anyof the above polynucleotide sequences, and a polynucleotide sequencecomprising all or a fragment of any of such polynucleotide sequenceswherein the sequence preferably encodes a hemagglutinin or neuraminidasepolypeptide or a fragment of a hemagglutinin or neuraminidasepolypeptide. The invention also includes an isolated or recombinantnucleic acid that encodes an amino acid sequence which is substantiallyidentical over at least about 300 amino acids of any of the abovenucleic acids, or over at least about 350 amino acids; over at leastabout 400 amino acids; over at least about 450 amino acids; over atleast about 500 amino acids; over at least about 502 amino acids; overat least about 550 amino acids; over at least about 559 amino acids;over at least about 565 amino acids; or over at least about 566 aminoacids of any of the above nucleic acids. Again, in situations whereinthe amino acid is less than, e.g., 566, 565, 559, etc. in length (e.g.,see, Sequence Listing) then it should be understood that the length isoptionally less than 566, 565, 559, etc. The invention also includes anyof the above nucleic acids that comprise a hemagglutinin orneuraminidase polypeptide, or one or more fragments of one or morehemagglutinin or neuraminidase polypeptide. Other aspects of theinvention include isolated or recombinant nucleic acids that encode apolypeptide (optionally a hemagglutinin or neuraminidase polypeptide)whose sequence has at least 98% identity, at least 98.5% identity, atleast 99% identity, at least 99.2% identity, at least 99.4% identity, atleast 99.6% identity, at least 99.8% identity, or at least 99.9%identity to at least one of the above described polynucleotides. Theinvention also includes isolated or recombinant nucleic acids encoding apolypeptide of hemagglutinin or neuraminidase produced by mutating orrecombining one or more above described polynucleotide sequences. Thepolynucleotide sequences of the invention can optionally comprise one ormore of, e.g., a leader sequence, a precursor sequence, or an epitopetag sequence or the like, and can optionally encode a fusion protein(e.g., with one or more additional nucleic acid sequences).

In yet other embodiments, the invention comprises a composition ofmatter having two or more above described nucleic acids (e.g., a librarycomprising at least about 2, 5, 10, 50 or more nucleic acids). Suchcompositions can optionally be produced by cleaving one or more abovedescribed nucleic acid (e.g., mechanically, chemically, enzymaticallywith a restriction endonuclease/RNAse/DNAse, etc.). Other compositionsof the invention include, e.g., compositions produced by incubating oneor more above described nucleic acid in the presence ofdeoxyribonucleotide triphosphates and a thermostable nucleic acidpolymerase.

The invention also encompasses cells comprising at least one of theabove described nucleic acids, or a cleaved or amplified fragment orproduct thereof. Such cells can optionally express a polypeptide encodedby such nucleic acid. Other embodiments of the invention include vectors(e.g., plasmids, cosmids, phage, viruses, virus fragments, etc.)comprising any of above described nucleic acids. Such vectors canoptionally comprise an expression vector. Preferred expression vectorsof the invention include, but are not limited to, vectors comprising polI promoter and terminator sequences or vectors using both the pol I andpol II promoters “the poll/polIl promoter system” (e.g., Zobel et al.,Nucl. Acids Res. 1993, 21:3607; US20020164770; Neumann et al., Proc.Natl. Acad. Sci. USA 1999, 96:9345; Fodor et al., J. Virol. 1999,73:9679; and US20030035814). Cells transduced by such vectors are alsowithin the current invention.

In some embodiments, the invention encompasses a virus (e.g., aninfluenza virus) comprising one or more above described nucleic acids(e.g., encoding hemagglutinin and/or neuraminidase), or one or morefragments thereof. Immunogenic compositions comprising such virus arealso part of the current invention. Such viruses can comprises areassortment virus such as a 6:2 reassortment virus (e.g., comprising 6gene encoding regions from one or more donor virus and 2 gene encodingregions from one or more above described nucleotide sequence (or one ormore fragment thereof) which can optionally comprise hemagglutininand/or neuraminidase). Reassortment viruses (optionally live viruses) ofthe invention can include donor viruses that are one or more of, e.g.,cold-sensitive, cold-adapted, or an attenuated. For example,reassortment viruses can comprise e.g., A/Ann Arbor/6/60, PR8, etc.Reassortment viruses of the invention may alternatively exclude A/AnnArbor/6/60. One preferred embodiment of the invention is a reassortantinfluenza virus, wherein the virus is a 6:2 reassortment influenza virusand comprises 6 gene encoding regions from A/Ann Arbor/6/60 and 2 geneencoding regions that encode a polypeptide selected from the groupconsisting of: the polypeptides of SEQ ID NOS:11-20. In an alternativeembodiment, a reassortant influenza virus of the invention includes a6:2 reassortment influenza virus, wherein said virus comprises 6 geneencoding regions from one or more donor viruses other than A/AnnArbor/6/60 and 2 gene encoding regions that encode a polypeptideselected from the group consisting of: the polypeptides of SEQ IDNOS:11-20. In another alternative embodiment, a reassortant influenzavirus of the invention includes a 6:2 reassortment influenza virus,wherein said virus comprises 6 gene encoding regions from one or moredonor viruses other than A/Ann Arbor/6/60 and 2 gene encoding regions,wherein the 2 gene encoding regions are HA or NA polypeptides from anypandemic influenza strain. Methods of producing recombinant influenzavirus through culturing a host cell harboring an influenza virus in asuitable culture medium under conditions permitting expression ofnucleic acid and, isolating the recombinant influenza virus from one ormore of the host cell or the medium are also part of the invention.

In other embodiments herein, the invention comprises immunogeniccompositions having an immunologically effective amount of any of theabove described recombinant influenza virus. Other embodiments includemethods for stimulating the immune system of an individual to produce aprotective immune response against influenza virus by administering tothe individual an immunologically effective amount of any of therecombinant influenza virus described above (optionally in aphysiologically effective carrier).

Other aspects of the invention include methods of producing an isolatedor recombinant polypeptide by culturing any host cell above, in asuitable culture medium under conditions permitting expression ofnucleic acid and, isolating the polypeptide from one or more of the hostcells or the medium in which is the cells are grown.

Immunogenic compositions are also features of the invention. Forexample, immunogenic compositions comprising one or more of any of thepolypeptides and/or nucleic acids described above and, optionally, anexcipient such as a pharmaceutically acceptable excipient or one or morepharmaceutically acceptable administration component. Immunogeniccompositions of the invention can also comprise any one or more abovedescribed virus as well (e.g., along with one or more pharmaceuticallyacceptable administration component).

Methods of producing immunogenic responses in a subject throughadministration of an effective amount of any of the above viruses (orimmunogenic compositions) to a subject are also within the currentinvention. Additionally, methods of prophylactic or therapeutictreatment of a viral infection (e.g., viral influenza) in a subjectthrough administration of any one or more above described virus (orimmunogenic compositions) in an amount effective to produce animmunogenic response against the viral infection are also part of thecurrent invention. Subjects for such treatment can include mammals(e.g., humans). Such methods can also comprise in vivo administration tothe subject as well as in vitro or ex vivo administration to one or morecells of the subject. Additionally, such methods can also compriseadministration of a composition of the virus and a pharmaceuticallyacceptable excipient that are administered to the subject in an amounteffect to prophylactically or therapeutically treat the viral infection.

In other aspects the invention includes compositions of mattercomprising nucleic acid sequences encoding hemagglutinin and/orneuraminidase polypeptides of one or more pandemic influenza strain andnucleic acid sequences encoding one or more polypeptide of A/AnnArbor/6/60. Additionally, the invention includes compositions of mattercomprising nucleic acid sequences encoding hemagglutinin and/orneuraminidase polypeptides of one or more pandemic influenza strain andnucleic acid sequences encoding one or more polypeptide of PR8 or A/AnnArbor/6/60. Such sequences can include those listed in the SequenceListing herein. Additionally, preferred embodiments of the inventioninclude compositions of matter comprising sequences encodinghemagglutinin and/or neuraminidase of one or more pandemic influenzastrain and nucleic acid sequences encoding a selected backbone strain ina 6:2 reassortment. Such compositions preferably include sequencesencoding the hemagglutinin and neuraminidase selected from the SequenceListing herein and a backbone strain, wherein the backbone strain is PR8or A/Ann Arbor/6/60. The invention also includes such compositions asdescribed above wherein the hemagglutinin comprises a modified polybasiccleavage site. The invention also includes live attenuated influenzavaccine comprising such above compositions.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying figures and appendix.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows modifications engineered into the HA gene of VN/1203/2004to remove the polybasic cleavage site.

FIG. 2: Displays results showing that intranasally administered H5N1 careassortant viruses do not replicate in chickens.

FIG. 3: Illustrates that the H5N1/AA ca vaccine candidates are notlethal to mice.

FIG. 4: Illustrates that the 1997 and 2004H5N1 ca reassortant virusesare restricted in replication in mice.

FIG. 5: Illustrates that the reassortant H5N1/AA ca influenza virusesare restricted in replication in lungs of mice.

FIG. 6: Shows the serum HAI Ab titers elicited in mice following asingle i.n. dose of vaccine.

FIG. 7: Shows serum neutralizing Ab titers elicited in mice following asingle i.n. dose of vaccine.

FIG. 8: Illustrates that H5N1 ca reassortant viruses protect mice fromlethal challenges with 50, 500 or 5000 LD₅₀ of wild-type H5N1 viruses.

FIG. 9: Illustrates the efficacy of protection from pulmonaryreplication of homologous and heterologous H5N1 challenge viruses inmice.

FIG. 10: Illustrates the efficacy of protection from replication ofhomologous and heterologous H5N1 challenge viruses in the upperrespiratory tract of mice.

FIG. 11: Illustrates the efficacy of protection conferred by 2004H5N1 cavaccine against high dose (10₅TCID₅₀) challenge with homologous orheterologous H5N1 wt viruses in mice.

FIG. 12: Illustrates the efficacy of protection conferred by 1997 and2003H5N1 ca vaccines against high dose (10₅TCID₅₀) challenges withhomologous or heterologous H5N1 wild-type viruses in mice.

FIG. 13: Illustrates the efficacy of protection conferred by 2004H5N1 cavaccine against low or high doses of homologous H5N1 wild-type viruschallenges in mice.

DETAILED DESCRIPTION

The present invention includes polypeptide and polynucleotide sequencesof influenza hemagglutinin and neuraminidase as well as vectors,compositions and the like comprising such sequences and methods of theiruse. Additional features of the invention are described in more detailherein.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. The following definitionssupplement those in the art and are directed to the current applicationand are not necessarily to be imputed to any related or unrelated case,e.g., to any commonly owned patent or application. Although any methodsand materials similar or equivalent to those described herein can beused in the practice for testing of the present invention, the preferredmaterials and methods are described herein. Accordingly, the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a virus”includes a plurality of viruses; reference to a “host cell” includesmixtures of host cells, and the like.

An “amino acid sequence” is a polymer of amino acid residues (a protein,polypeptide, etc.) or a character string representing an amino acidpolymer, depending on context.

The terms “nucleic acid,” “polynucleotide,” “polynucleotide sequence”and “nucleic acid sequence” refer to single-stranded or double-strandeddeoxyribonucleotide or ribonucleotide polymers, chimeras or analoguesthereof, or a character string representing such, depending on context.As used herein, the term optionally includes polymers of analogs ofnaturally occurring nucleotides having the essential nature of naturalnucleotides in that they hybridize to single-stranded nucleic acids in amanner similar to naturally occurring nucleotides (e.g., peptide nucleicacids). Unless otherwise indicated, a particular nucleic acid sequenceof this invention optionally encompasses complementary sequences inaddition to the sequence explicitly indicated. From any specifiedpolynucleotide sequence, either the given nucleic acid or thecomplementary polynucleotide sequence (e.g., the complementary nucleicacid) can be determined.

The term “nucleic acid” or “polynucleotide” also encompasses anyphysical string of monomer units that can be corresponded to a string ofnucleotides, including a polymer of nucleotides (e.g., a typical DNA orRNA polymer), PNAs, modified oligonucleotides (e.g., oligonucleotidescomprising bases that are not typical to biological RNA or DNA insolution, such as 2′-O-methylated oligonucleotides), and the like. Anucleic acid can be e.g., single-stranded or double-stranded.

A “subsequence” is any portion of an entire sequence, up to andincluding the complete sequence. Typically, a subsequence comprises lessthan the full-length sequence. A “unique subsequence” is a subsequencethat is not found in any previously determined influenza polynucleotideor polypeptide sequence

The term “variant” with respect to a polypeptide refers to an amino acidsequence that is altered by one or more amino acids with respect to areference sequence. The variant can have “conservative” changes, whereina substituted amino acid has similar structural or chemical properties,e.g., replacement of leucine with isoleucine. Alternatively, a variantcan have “nonconservative” changes, e.g., replacement of a glycine witha tryptophan. Analogous minor variation can also include amino aciddeletion or insertion, or both. Guidance in determining which amino acidresidues can be substituted, inserted, or deleted without eliminatingbiological or immunological activity can be found using computerprograms well known in the art, for example, DNASTAR software. Examplesof conservative substitutions are also described herein.

The term “gene” is used broadly to refer to any nucleic acid associatedwith a biological function. Thus, genes include coding sequences and/orthe regulatory sequences required for their expression. The term “gene”applies to a specific genomic sequence, as well as to a cDNA or an mRNAencoded by that genomic sequence.

Genes also include non-expressed nucleic acid segments that, forexample, form recognition sequences for other proteins. Non-expressedregulatory sequences include “promoters” and “enhancers,” to whichregulatory proteins such as transcription factors bind, resulting intranscription of adjacent or nearby sequences. A “tissue specific”promoter or enhancer is one that regulates transcription in a specifictissue type or cell type, or types.

“Expression of a gene” or “expression of a nucleic acid” meanstranscription of DNA into RNA (optionally including modification of theRNA, e.g., splicing), translation of RNA into a polypeptide (possiblyincluding subsequent modification of the polypeptide, e.g.,post-translational modification), or both transcription and translation,as indicated by the context.

An “open reading frame” or “ORF” is a possible translational readingframe of DNA or RNA (e.g., of a gene), which is capable of beingtranslated into a polypeptide. That is, the reading frame is notinterrupted by stop codons. However, it should be noted that the termORF does not necessarily indicate that the polynucleotide is, in fact,translated into a polypeptide.

The term “vector” refers to the means by which a nucleic acid can bepropagated and/or transferred between organisms, cells, or cellularcomponents. Vectors include plasmids, viruses, bacteriophages,pro-viruses, phagemids, transposons, artificial chromosomes, and thelike, that replicate autonomously or can integrate into a chromosome ofa host cell. A vector can also be a naked RNA polynucleotide, a nakedDNA polynucleotide, a polynucleotide composed of both DNA and RNA withinthe same strand, a poly-lysine-conjugated DNA or RNA, apeptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like,that is not autonomously replicating. In many, but not all, commonembodiments, the vectors of the present invention are plasmids.

An “expression vector” is a vector, such as a plasmid that is capable ofpromoting expression, as well as replication of a nucleic acidincorporated therein. Typically, the nucleic acid to be expressed is“operably linked” to a promoter and/or enhancer, and is subject totranscription regulatory control by the promoter and/or enhancer.

A “bi-directional expression vector” is characterized by two alternativepromoters oriented in the opposite direction relative to a nucleic acidsituated between the two promoters, such that expression can beinitiated in both orientations resulting in, e.g., transcription of bothplus (+) or sense strand, and negative (−) or antisense strand RNAs.

A “polypeptide” is a polymer comprising two or more amino acid residues(e.g., a peptide or a protein). The polymer can optionally comprisemodifications such as glycosylation or the like. The amino acid residuesof the polypeptide can be natural or non-natural and can beunsubstituted, unmodified, substituted or modified.

In the context of the invention, the term “isolated” refers to abiological material, such as a virus, a nucleic acid or a protein, whichis substantially free from components that normally accompany orinteract with it in its naturally occurring environment. The isolatedbiological material optionally comprises additional material not foundwith the biological material in its natural environment, e.g., a cell orwild-type virus. For example, if the material is in its naturalenvironment, such as a cell, the material can have been placed at alocation in the cell (e.g., genome or genetic element) not native tosuch material found in that environment. For example, a naturallyoccurring nucleic acid (e.g., a coding sequence, a promoter, anenhancer, etc.) becomes isolated if it is introduced by non-naturallyoccurring means to a locus of the genome (e.g., a vector, such as aplasmid or virus vector, or amplicon) not native to that nucleic acid.Such nucleic acids are also referred to as “heterologous” nucleic acids.An isolated virus, for example, is in an environment (e.g., a cellculture system, or purified from cell culture) other than the nativeenvironment of wild-type virus (e.g., the nasopharynx of an infectedindividual).

The term “chimeric” or “chimera,” when referring to a virus, indicatesthat the virus includes genetic and/or polypeptide components derivedfrom more than one parental viral strain or source. Similarly, the term“chimeric” or “chimera,” when referring to a viral protein, indicatesthat the protein includes polypeptide components (i.e., amino acidsubsequences) derived from more than one parental viral strain orsource.

The term “recombinant” indicates that the material (e.g., a nucleic acidor protein) has been artificially or synthetically (non-naturally)altered by human intervention. The alteration can be performed on thematerial within, or removed from, its natural environment or state.Specifically, e.g., an influenza virus is recombinant when it isproduced by the expression of a recombinant nucleic acid. For example, a“recombinant nucleic acid” is one that is made by recombining nucleicacids, e.g., during cloning, DNA shuffling or other procedures, or bychemical or other mutagenesis; a “recombinant polypeptide” or“recombinant protein” is a polypeptide or protein which is produced byexpression of a recombinant nucleic acid; and a “recombinant virus”,e.g., a recombinant influenza virus, is produced by the expression of arecombinant nucleic acid.

The term “reassortant,” when referring to a virus, indicates that thevirus includes genetic and/or polypeptide components derived from morethan one parental viral strain or source. For example, a 7:1 reassortantincludes 7 viral genomic segments (or gene segments) derived from afirst parental virus, and a single complementary viral genomic segment,e.g., encoding a hemagglutinin or neuraminidase of the invention. A 6:2reassortant includes 6 genomic segments, most commonly the 6 internalgenes from a first parental virus, and two complementary segments, e.g.,hemagglutinin and neuraminidase, from a different parental virus.

The term “introduced” when referring to a heterologous or isolatednucleic acid refers to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid can beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA). The term includes suchmethods as “infection,” “transfection,” “transformation” and“transduction.” In the context of the invention a variety of methods canbe employed to introduce nucleic acids into cells, includingelectroporation, calcium phosphate precipitation, lipid mediatedtransfection (lipofection), etc.

The term “host cell” means a cell that contains a heterologous nucleicacid, such as a vector, and supports the replication and/or expressionof the nucleic acid. Host cells can be prokaryotic cells such as E.coli, or eukaryotic cells such as yeast, insect, amphibian, avian ormammalian cells, including human cells. Exemplary host cells caninclude, e.g., Vero (African green monkey kidney) cells, BHK (babyhamster kidney) cells, primary chick kidney (PCK) cells, Madin-DarbyCanine Kidney (MDCK) cells, Madin-Darby Bovine Kidney (MDBK) cells, 293cells (e.g., 293T cells), and COS cells (e.g., COS1, COS7 cells), etc.

An “immunologically effective amount” of influenza virus is an amountsufficient to enhance an individual's (e.g., a human's) own immuneresponse against a subsequent exposure to influenza virus. Levels ofinduced immunity can be monitored, e.g., by measuring amounts ofneutralizing secretory and/or serum antibodies, e.g., by plaqueneutralization, complement fixation, enzyme-linked immunosorbent, ormicroneutralization assay.

A “protective immune response” against influenza virus refers to animmune response exhibited by an individual (e.g., a human) that isprotective against disease when the individual is subsequently exposedto and/or infected with such influenza virus. In some instances, theinfluenza virus (e.g., naturally circulating) can still cause infection,but it cannot cause a serious infection. Typically, the protectiveimmune response results in detectable levels of host engendered serumand secretory antibodies that are capable of neutralizing virus of thesame strain and/or subgroup (and possibly also of a different,non-vaccine strain and/or subgroup) in vitro and in vivo.

As used herein, an “antibody” is a protein comprising one or morepolypeptides substantially or partially encoded by immunoglobulin genesor fragments of immunoglobulin genes. The recognized immunoglobulingenes include the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes, as well as myriad immunoglobulin variable regiongenes. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. A typical immunoglobulin (antibody) structural unitcomprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one “light” (about 25 kD) andone “heavy” chain (about 50-70 kD). The N-terminus of each chain definesa variable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively. Antibodies exist as intact immunoglobulins or as a numberof well-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′2, a dimer ofFab which itself is a light chain joined to VH-CH1 by a disulfide bond.The F(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into aFab′ monomer. The Fab′ monomer is essentially a Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1999), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein, includes antibodies or fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Antibodies include, e.g., polyclonalantibodies, monoclonal antibodies, multiple or single chain antibodies,including single chain Fv (sFv or scFv) antibodies in which a variableheavy and a variable light chain are joined together (directly orthrough a peptide linker) to form a continuous polypeptide, andhumanized or chimeric antibodies.

Influenza Virus

The polypeptides and polynucleotides of the invention, e.g., SEQ ID NO:1-20, are variants of influenza HA and NA sequences. In general,influenza viruses are made up of an internal ribonucleoprotein corecontaining a segmented single-stranded RNA genome and an outerlipoprotein envelope lined by a matrix protein. The genome of influenzaviruses is composed of eight segments of linear (−) strand ribonucleicacid (RNA), encoding the immunogenic hemagglutinin (HA) andneuraminidase (NA) proteins, and six internal core polypeptides: thenucleocapsid nucleoprotein (NP); matrix proteins (M); non-structuralproteins (NS); and 3 RNA polymerase (PA, PB1, PB2) proteins. Duringreplication, the genomic viral RNA is transcribed into (+) strandmessenger RNA and (−) strand genomic cRNA in the nucleus of the hostcell. Each of the eight genomic segments is packaged intoribonucleoprotein complexes that contain, in addition to the RNA, NP anda polymerase complex (PB1, PB2, and PA).

Influenza is commonly grouped into influenza A and influenza Bcategories. Influenza A and influenza B viruses each contain eightsegments of single stranded RNA with negative polarity. The influenza Agenome encodes eleven polypeptides. Segments 1-3 encode threepolypeptides, making up a RNA-dependent RNA polymerase. Segment 1encodes the polymerase complex protein PB2. The remaining polymeraseproteins PB1 and PA are encoded by segment 2 and segment 3,respectively. In addition, segment 1 of some influenza strains encodes asmall protein, PB1-F2, produced from an alternative reading frame withinthe PB1 coding region. Segment 4 encodes the hemagglutinin (HA) surfaceglycoprotein involved in cell attachment and entry during infection.Segment 5 encodes the nucleocapsid nucleoprotein (NP) polypeptide, themajor structural component associated with viral RNA. Segment 6 encodesa neuraminidase (NA) envelope glycoprotein. Segment 7 encodes two matrixproteins, designated M1 and M2, which are translated from differentiallyspliced mRNAs. Segment 8 encodes NS1 and NS2, two nonstructuralproteins, which are translated from alternatively spliced mRNA variants.The eight genome segments of influenza B encode 11 proteins. The threelargest genes code for components of the RNA polymerase, PB1, PB2 andPA. Segment 4 encodes the HA protein. Segment 5 encodes NP. Segment 6encodes the NA protein and the NB protein. Both proteins, NB and NA, aretranslated from overlapping reading frames of a bicistronic mRNA.Segment 7 of influenza B also encodes two proteins: M1 and BM2. Thesmallest segment encodes two products: NS1 is translated from the fulllength RNA, while NS2 is translated from a spliced mRNA variant.

Influenza Virus Vaccines

The sequences, compositions and methods herein are primarily, but notsolely, concerned with production of influenza viruses for vaccines.Historically, influenza virus vaccines have primarily been produced inembryonated hen eggs using strains of virus selected or based onempirical predictions of relevant strains. More recently, reassortantviruses have been produced that incorporate selected hemagglutininand/or neuraminidase antigens in the context of an approved attenuated,temperature sensitive master strain. Following culture of the virusthrough multiple passages in hen eggs, influenza viruses are recoveredand, optionally, inactivated, e.g., using formaldehyde and/orβ-propiolactone (or alternatively used in live attenuated vaccines).Thus, it will be appreciated that HA and NA sequences (e.g., SEQ ID NO:1-20) are quite useful in constructing influenza vaccines. The currentinvention includes viruses/vaccines comprising HA and/or NA sequences ofpandemic influenza strains (including wherein the HA sequences comprisemodified polybasic cleavage sites such as the modifications describedherein); and including wherein the viruses/vaccines comprise a cabackbone such as A/AA/6/60 or the backbone of PR8.

Attempts at producing recombinant and reassortant vaccines in cellculture have been hampered by the inability of some of the strainsapproved for vaccine production to grow efficiently under standard cellculture conditions. However, prior work by the inventors and theircoworkers provided a vector system, and methods for producingrecombinant and reassortant viruses in culture, thus, making it possibleto rapidly produce vaccines corresponding to one or many selectedantigenic strains of virus, e.g., either A or B strains, varioussubtypes or substrains, etc., e.g., comprising the HA and/or NAsequences herein. See, Multi-Plasmid System for the production ofInfluenza virus, U.S. Application No. 60/420,708, filed Oct. 23, 2002,U.S. application Ser. No. 10/423,828, filed Apr. 25, 2003 and U.S.Application 60/574,117 filed May 24, 2004. Typically, the cultures aremaintained in a system, such as a cell culture incubator, undercontrolled humidity and CO₂, at constant temperature using a temperatureregulator, such as a thermostat to insure that the temperature does notexceed 35° C. Reassortant influenza viruses can be readily obtained byintroducing a subset of vectors corresponding to genomic segments of amaster influenza virus, in combination with complementary segmentsderived from strains of interest (e.g., HA and/or NA antigenic variantsherein). Typically, the master strains are selected on the basis ofdesirable properties relevant to vaccine administration. For example,for vaccine production, e.g., for production of a live attenuatedvaccine, the master donor virus strain may be selected for an attenuatedphenotype, cold adaptation and/or temperature sensitivity. As explainedelsewhere herein and, e.g., in U.S. patent application Ser. No.10/423,828, etc., various embodiments of the invention utilize A/AnnArbor (AA)/6/60 influenza strain as a “backbone” upon which to add HAand/or NA genes (e.g., such as those sequences listed herein, etc.) tocreate desired reassortant viruses. Thus, for example, in a 6:2reassortant, 2 genes (i.e., NA and HA) would be from the influenzastrain(s) against which an immunogenic reaction is desired, while theother 6 genes would be from the Ann Arbor strain, or other backbonestrain, etc. The Ann Arbor virus is useful for its cold adapted,attenuated, temperature sensitive attributes. Of course, it will beappreciated that the HA and NA sequences herein are capable ofreassortment with a number of other virus genes or virus types (e.g., anumber of different “backbones” such as PR8, etc., containing the otherinfluenza genes present in a reassortant, namely, the non-HA and non-NAgenes

Various embodiments herein can comprise live attenuated vaccines, havingthe HA and/or NA sequences herein, for pandemic influenza. Such vaccinestypically comprise, e.g., the HA and/or NA sequences of SEQ ID NO:11-20, or their corresponding nucleotides of SEQ ID NO: 1-10. Oneproblem arising from growth of vaccine virus strains (e.g.,reassortants) in eggs is that avian strains (which can be involved inpandemics) can kill the eggs in which the vaccines are to be producedand are, thus, hard to manipulate, produce, etc. through use oftraditional (non-plasmid rescue) reassortant production. Such avianstrains are of interest since evidence indicates they can result ininfluenza in humans and possible pandemics. Thus, use of plasmid-rescuesystems to create/manipulate influenza reassortants with pandemicstrains such as various avian sequences (e.g., the HA and NA sequencesherein) are quite desirable and are features of the invention. It willbe appreciated, however, that the current sequences are also capable ofuse with non-plasmid or traditional systems.

Aquatic birds (among others) can be infected by influenza A viruses of15 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes. Such birds canserve as a reservoir from which novel influenza subtypes can beintroduced into human populations and cause pandemics. The observationthat avian H7N7 influenza A viruses infected humans in The Netherlandsin 2003 and avian H5N1 and H9N2 viruses infected humans in Hong Kong andChina earlier, raise concerns that these (and other) subtypes have thepotential to cause pandemics. Thus, vaccines are needed to prevent humaninfections with avian influenza A viruses. Live, attenuated influenza Avirus vaccines against human influenza viruses were recently licensed inthe United States. See above. Such vaccines are reassortant H1N1 andH3N2 viruses in which the internal protein genes of A/Ann Arbor(AA)/6/60 (H2N2) cold adapted (ca) virus confer the cold adapted,attenuation and temperature sensitive phenotypes of the AA ca virus onthe reassortant viruses (i.e., the ones having the hemagglutinin andneuraminidase genes from the non-Ann Arbor strain). Classical geneticreassortment and plasmid-based reverse genetics techniques have beenapplied to generate reassortant viruses that contain the hemagglutininand neuraminidase genes from avian influenza A viruses (H4-H14 subtypes)and six internal gene segments from the AA ca virus. Such reassortantviruses are features of the invention. See the HA and NA gene sequencesbelow. These viruses bear biological properties that are desirable incandidate vaccines because the phenotypes associated with the AA cavirus are present in the reassortant viruses. The generation andevaluation of these reassortant viruses as seed viruses for vaccines areimportant steps in pandemic preparedness. It is contemplated thatclinical trials can establish the safety, infectivity and immunogenicityof such live attenuated pandemic vaccines. Other embodiments of theinvention include reassortant viruses (e.g., those used in vaccines)comprising pandemic antigenic genes HA and/or NA from, e.g., avian,porcine, etc., pandemic virus strains in addition to those listedherein, to produce pandemic vaccines which are created throughplasmid-rescue reassortment (e.g., reassortment with A/Ann Arbor 6/60(i.e., A/AA/6/60), PR8, etc. Methods of construction and use of suchviruses and vaccines are also included. “Pandemic virus strains” as usedherein is defined as an influenza strain A virus subtype that it is notcirculating in the human population, that is declared to be a pandemicstrain by the Centers for Disease Control or the World HealthOrganization or generally acknowledged as such within the scientificcommunity.

In various embodiments herein, the antigenic sequences (e.g., the HAsequences) as well as viruses and vaccines from such viruses comprisemodified polybasic cleavage sites. Some highly pathogenic avian pandemicinfluenza strains comprise multiple basic amino acid cleavage siteswithin hemagglutinin sequences. See, e.g., Li et al., J. of InfectiousDiseases, 179:1132-8, 1999. Such cleavage sites, in typical embodimentsherein, are, e.g., modified or altered in their sequences in comparisonto the wild-type sequences from which the current sequences are derived(e.g., to disable the cleavage or reduce the cleavage there, etc.). Suchmodifications/alterations can be different in different strains due tothe various sequences of the cleavage sites in the wild-type sequences.For example, 4 polybasic residues (RRKK) at 326-329 of mature H5 aretypically removed in sequences herein (as compared to wt). See sequencelisting and FIG. 1. In various embodiments, the polybasic cleavage sitescan be modified in a number of ways (all of which are contained withinthe invention). For example, the polybasic cleavage site can be removedone amino acid at a time (e.g., one R removed, two R5 removed, RRKremoved, or RRKK removed). Additionally, the amino acid residue directlyupstream of the cleavage site can also be removed or altered (e.g., froman R to a T, etc.); also, the nucleotides encoding the amino acidresidue directly after the cleavage site can also be modified. See,e.g., FIG. 1 for an illustration of cleavage site modification. Inaddition, hemagglutinin polypeptide sequences of influenza viruscomprise amino terminal signal peptide sequences, thus, thehemagglutinin polypeptide sequences of the invention include both themature (amino terminal signal peptide cleaved) form of the hemagglutininpolypeptides and the pre-cleaved form of hemagglutinin. The cleavagesites of any hemagglutinin polypeptide sequence of any influenza straincan be routinely measured or predicted using any number of methods inthe art.

The terms “temperature sensitive,” “cold adapted” and “attenuated” asapplied to viruses (typically used as vaccines or for vaccineproduction) which optionally encompass the current sequences, are wellknown in the art. For example, the term “temperature sensitive” (ts)indicates, e.g., that the virus exhibits a 100 fold or greater reductionin titer at 39° C. relative to 33° C. for influenza A strains, or thatthe virus exhibits a 100 fold or greater reduction in titer at 37° C.relative to 33° C. for influenza B strains. The term “cold adapted” (ca)indicates that the virus exhibits growth at 25° C. within 100 fold ofits growth at 33° C., while the term “attenuated” (att) indicates thatthe virus replicates in the upper airways of ferrets but is notdetectable in their lung tissues, and does not cause influenza-likeillness in the animal. It will be understood that viruses withintermediate phenotypes, i.e., viruses exhibiting titer reductions lessthan 100 fold at 39° C. (for A strain viruses) or 37° C. (for B strainviruses), or exhibiting growth at 25° C. that is more than 100 fold thanits growth at 33° C. (e.g., within 200 fold, 500 fold, 1000 fold, 10,000fold less), and/or exhibit reduced growth in the lungs relative togrowth in the upper airways of ferrets (i.e., partially attenuated)and/or reduced influenza like illness in the animal, are also usefulviruses and can be used in conjunction with the HA and NA sequencesherein.

Again, the HA and NA sequences of the current invention are optionallyutilized in such plasmid reassortment vaccines (and/or in other ts, cs,ca, and/or att viruses and vaccines). However, it should be noted thatthe HA and NA sequences, etc. of the invention are not limited tospecific vaccine compositions or production methods, and can, thus, beutilized in substantially any vaccine type or vaccine production methodwhich utilizes strain specific HA and NA antigens (e.g., any of SEQ IDNO: 11-20 or the corresponding nucleotides in SEQ ID NO: 1-10).

FluMist™

As mentioned previously, numerous examples and types of influenzavaccine exist. An exemplary influenza vaccine is FluMist™ which is alive, attenuated vaccine that protects children and adults frominfluenza illness (Belshe et al. (1998) The efficacy of live attenuated,cold-adapted, trivalent, intranasal influenza virus vaccine in childrenN Engl J Med 338:1405-12; Nichol et al. (1999) Effectiveness of live,attenuated intranasal influenza virus vaccine in healthy, workingadults: a randomized controlled trial JAMA 282:137-44). In typical, andpreferred, embodiments, the methods and compositions of the currentinvention are preferably adapted to/used with production of FluMist™vaccine. However, it will be appreciated by those skilled in the artthat the sequences, methods, compositions, etc. herein are alsoadaptable to production of similar or even different viral vaccines.

FluMist™ vaccine strains contain, e.g., HA and NA gene segments derivedfrom the strains (e.g., wild-type strains) to which the vaccine isaddressed along with six gene segments, PB1, PB2, PA, NP, M and NS, froma common master donor virus (MDV). The HA sequences herein, thus, arepart of various FluMist™ formulations. The MDV for influenza A strainsof FluMist™ (MDV-A), was created by serial passage of the wild-typeA/Ann Arbor/6/60 (A/AA/6/60) strain in primary chicken kidney tissueculture at successively lower temperatures (Maassab (1967) Adaptationand growth characteristics of influenza virus at 25 degrees C. Nature213:612-4). MDV-A replicates efficiently at 25° C. (ca, cold adapted),but its growth is restricted at 38 and 39° C. (ts, temperaturesensitive). Additionally, this virus does not replicate in the lungs ofinfected ferrets (att, attenuation). The ts phenotype is believed tocontribute to the attenuation of the vaccine in humans by restrictingits replication in all but the coolest regions of the respiratory tract.The stability of this property has been demonstrated in animal modelsand clinical studies. In contrast to the ts phenotype of influenzastrains created by chemical mutagenesis, the ts property of MDV-A doesnot revert following passage through infected hamsters or in shedisolates from children (for a recent review, see Murphy & Coelingh(2002) Principles underlying the development and use of live attenuatedcold-adapted influenza A and B virus vaccines Viral Immunol 15:295-323).

Clinical studies in over 20,000 adults and children involving 12separate 6:2 reassortant strains have shown that these vaccines areattenuated, safe and efficacious (Belshe et al. (1998) The efficacy oflive attenuated, cold-adapted, trivalent, intranasal influenza virusvaccine in children N Engl J Med 338:1405-12; Boyce et al. (2000) Safetyand immunogenicity of adjuvanted and unadjuvanted subunit influenzavaccines administered intranasally to healthy adults Vaccine 19:217-26;Edwards et al. (1994) A randomized controlled trial of cold adapted andinactivated vaccines for the prevention of influenza A disease J InfectDis 169:68-76; Nichol et al. (1999) Effectiveness of live, attenuatedintranasal influenza virus vaccine in healthy, working adults: arandomized controlled trial JAMA 282:137-44). Reassortants carrying thesix internal genes of MDV-A and the two HA and NA gene segments of awild-type virus (i.e., a 6:2 reassortant) consistently maintain ca, tsand att phenotypes (Maassab et al. (1982) Evaluation of acold-recombinant influenza virus vaccine in ferrets J. Infect. Dis.146:780-900).

Production of such reassorted virus using B strains of influenza is moredifficult, however, recent work (see, e.g., Multi-Plasmid System for theProduction of Influenza Virus, U.S. Application No. 60/420,708, filedOct. 23, 2002, U.S. application Ser. No. 10/423,828, filed Apr. 25,2003, and U.S. Application No. 60/574,117, filed May 24, 2004) has shownan eight plasmid system for the generation of influenza B virus entirelyfrom cloned cDNA. Methods for the production of attenuated liveinfluenza A and B virus suitable for vaccine formulations, such as livevirus vaccine formulations useful for intranasal administration werealso shown.

The system and methods described previously are useful for the rapidproduction in cell culture of recombinant and reassortant influenza Aand B viruses, including viruses suitable for use as vaccines, includinglive attenuated vaccines, such as vaccines suitable for intranasaladministration. The sequences (e.g., SEQ ID NO: 1-10 and thecorresponding amino acids in SEQ ID NO: 11-20), methods, etc. of thecurrent invention, are optionally used in conjunction with, or incombination with, such previous work involving, e.g., reassortedinfluenza viruses for vaccine production to produce viruses forvaccines.

Methods and Compositions for Prophylactic Administration of Vaccines

As stated above, alternatively, or in addition to, use in production ofFluMist™ vaccine, the current invention can be used in other vaccineformulations. In general, recombinant and reassortant viruses of theinvention (e.g., those comprising polynucleotides of SEQ ID NO:1-10 orpolypeptides of SEQ ID NO:11-20, or fragments thereof) can beadministered prophylactically in an immunologically effective amount andin an appropriate carrier or excipient to stimulate an immune responsespecific for one or more strains of influenza virus as determined by theHA and/or NA sequence. Typically, the carrier or excipient is apharmaceutically acceptable carrier or excipient, such as sterile water,aqueous saline solution, aqueous buffered saline solutions, aqueousdextrose solutions, aqueous glycerol solutions, ethanol, or combinationsthereof. The preparation of such solutions insuring sterility, pH,isotonicity, and stability is effected according to protocolsestablished in the art. Generally, a carrier or excipient is selected tominimize allergic and other undesirable effects, and to suit theparticular route of administration, e.g., subcutaneous, intramuscular,intranasal, etc.

A related aspect of the invention provides methods for stimulating theimmune system of an individual to produce a protective immune responseagainst influenza virus. In the methods, an immunologically effectiveamount of a recombinant influenza virus (e.g., comprising an HA and/orNA molecule of the invention), an immunologically effective amount of apolypeptide of the invention, and/or an immunologically effective amountof a nucleic acid of the invention is administered to the individual ina physiologically acceptable carrier.

Generally, the influenza viruses of the invention are administered in aquantity sufficient to stimulate an immune response specific for one ormore strains of influenza virus (i.e., against the HA and/or NA strainsof the invention). Preferably, administration of the influenza viruseselicits a protective immune response to such strains. Dosages andmethods for eliciting a protective immune response against one or moreinfluenza strains are known to those of skill in the art. See, e.g.,U.S. Pat. No. 5,922,326; Wright et al., Infect. Immun 37:397-400 (1982);Kim et al., Pediatrics 52:56-63 (1973); and Wright et al., J. Pediatr.88:931-936 (1976). For example, influenza viruses are provided in therange of about 1-1000 HID₅₀ (human infectious dose), i.e., about 10⁵-10⁸pfu (plaque forming units) per dose administered. Typically, the dosewill be adjusted within this range based on, e.g., age, physicalcondition, body weight, sex, diet, time of administration, and otherclinical factors. The prophylactic vaccine formulation is systemicallyadministered, e.g., by subcutaneous or intramuscular injection using aneedle and syringe, or a needle-less injection device. Alternatively,the vaccine formulation is administered intranasally, either by drops,large particle aerosol (greater than about 10 microns), or spray intothe upper respiratory tract. While any of the above routes of deliveryresults in a protective systemic immune response, intranasaladministration confers the added benefit of eliciting mucosal immunityat the site of entry of the influenza virus. For intranasaladministration, attenuated live virus vaccines are often preferred,e.g., an attenuated, cold adapted and/or temperature sensitiverecombinant or reassortant influenza virus. See above. While stimulationof a protective immune response with a single dose is preferred,additional dosages can be administered, by the same or different route,to achieve the desired prophylactic effect.

Typically, the attenuated recombinant influenza of this invention asused in a vaccine is sufficiently attenuated such that symptoms ofinfection, or at least symptoms of serious infection, will not occur inmost individuals immunized (or otherwise infected) with the attenuatedinfluenza virus. In some instances, the attenuated influenza virus canstill be capable of producing symptoms of mild illness (e.g., mild upperrespiratory illness) and/or of dissemination to unvaccinatedindividuals. However, its virulence is sufficiently abrogated such thatsevere lower respiratory tract infections do not occur in the vaccinatedor incidental host.

Alternatively, an immune response can be stimulated by ex vivo or invivo targeting of dendritic cells with influenza viruses comprising thesequences herein. For example, proliferating dendritic cells are exposedto viruses in a sufficient amount and for a sufficient period of time topermit capture of the influenza antigens by the dendritic cells. Thecells are then transferred into a subject to be vaccinated by standardintravenous transplantation methods.

While stimulation of a protective immune response with a single dose ispreferred, additional dosages can be administered, by the same ordifferent route, to achieve the desired prophylactic effect. In neonatesand infants, for example, multiple administrations may be required toelicit sufficient levels of immunity. Administration can continue atintervals throughout childhood, as necessary to maintain sufficientlevels of protection against wild-type influenza infection. Similarly,adults who are particularly susceptible to repeated or serious influenzainfection, such as, for example, health care workers, day care workers,family members of young children, the elderly, and individuals withcompromised cardiopulmonary function may require multiple immunizationsto establish and/or maintain protective immune responses. Levels ofinduced immunity can be monitored, for example, by measuring amounts ofneutralizing secretory and serum antibodies, and dosages adjusted orvaccinations repeated as necessary to elicit and maintain desired levelsof protection.

Optionally, the formulation for prophylactic administration of theinfluenza viruses also contains one or more adjuvants for enhancing theimmune response to the influenza antigens. Suitable adjuvants include:complete Freund's adjuvant, incomplete Freund's adjuvant, saponin,mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil orhydrocarbon emulsions, bacille Calmette-Guerin (BCG), Corynebacteriumparvum, and the synthetic adjuvant QS-21.

If desired, prophylactic vaccine administration of influenza viruses canbe performed in conjunction with administration of one or moreimmunostimulatory molecules. Immunostimulatory molecules include variouscytokines, lymphokines and chemokines with immunostimulatory,immunopotentiating, and pro-inflammatory activities, such asinterleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growthfactors (e.g., granulocyte-macrophage (GM)-colony stimulating factor(CSF)); and other immunostimulatory molecules, such as macrophageinflammatory factor, Flt3 ligand, B7.1; B7.2, etc. The immunostimulatorymolecules can be administered in the same formulation as the influenzaviruses, or can be administered separately. Either the protein (e.g., anHA and/or NA polypeptide of the invention,e.g., any of SEQ ID NO: 11-20)or an expression vector comprising a nucleic acid (e.g., any of SEQ IDNO: 1-10) encoding the protein can be administered to produce animmunostimulatory effect.

The above described methods are useful for therapeutically and/orprophylactically treating a disease or disorder, typically influenza, byintroducing a vector of the invention comprising a heterologouspolynucleotide encoding a therapeutically or prophylactically effectiveHA and/or NA polypeptide (or peptide) or HA and/or NA RNA (e.g., anantisense RNA or ribozyme) into a population of target cells in vitro,ex vivo or in vivo. Typically, the polynucleotide encoding thepolypeptide (or peptide), or RNA, of interest is operably linked toappropriate regulatory sequences as described above in the sectionsentitled “Expression Vectors” and “Additional Expression Elements.”Optionally, more than one heterologous coding sequence is incorporatedinto a single vector or virus. For example, in addition to apolynucleotide encoding a therapeutically or prophylactically active HAand/or NA polypeptide or RNA, the vector can also include additionaltherapeutic or prophylactic polypeptides, e.g., antigens, co-stimulatorymolecules, cytokines, antibodies, etc., and/or markers, and the like.

Although vaccination of an individual with an attenuated influenza virusof a particular strain of a particular subgroup can inducecross-protection against influenza virus of different strains and/orsubgroups, cross-protection can be enhanced, if desired, by vaccinatingthe individual with attenuated influenza virus from at least twostrains, e.g., each of which represents a different subgroup.Additionally, vaccine combinations can optionally include mixes ofpandemic vaccines (e.g., those against pandemic influenza strains suchas various avian strains, see, e.g., the sequences herein, or otherpandemic strains) and non-pandemic strains. Vaccine mixtures (ormultiple vaccinations) can comprise components from human strains and/ornon-human influenza strains (e.g., avian and human, etc.). Similarly,the attenuated influenza virus vaccines of this invention can optionallybe combined with vaccines that induce protective immune responsesagainst other infectious agents.

Polynucleotides of the Invention

It is well known in the art that the HA and NA polynucleotide segmentsof influenza viruses comprise both a coding region (encoding the ORF)and noncoding regions (NCRs), both 5′ and 3′ of the HA and NA codingsequence. An example of these NCRs are shown in SEQ ID NOS:1-9 (outsideof the ORFS). It is also known that primers can be made to these NCRs tofacilitate amplification of the entire HA and NA segments of influenzavirus. (see, e.g., Hoffmann et al. Arch Virol. 2001 December;146(12):2275-89). Further, it is known that the NCRs of the HA and NA ofinfluenza may increase the efficiency of acheiving reassortants.Therefore, the polynucleotide sequences of these NCRs (includingfragments and variants (e.g., at least about 60%, or at least 70%, or atleast 80%, or at least 90%, or at least about 91% or at least about 92%,or at least about 93%, or at least about 94%, or at least about 95%, orat least about 96%, or at least about 97%, or at least about 98%, or atleast about 98.5%, or at least about 98.7%, or at least about 99%, or atleast about 99.1%, or at least about 99.2%, or at least about 99.3%, orat least about 99.4%, or at least about 99.5%, or at least about 99.6%or at least about 99.7%, or at least about 99.8%, or at least about99.9% identity) thereof) are within the scope of this invention. Whenamplifying the HA and NA segments of any pandemic strain, one could makeand use polynucleotide primers to bind conserved (e.g., among relatedstrains) regions of the HA and NA NCRs for amplification (e.g., byRT-PCR). In one embodiment, HA and NA polynucleotides of the inventioninclude both the NCR and ORF of the HA and NA sequences (includingfragments and variants (e.g., at least about 60%, or at least 70%, or atleast 80%, or at least 90%, or at least about 91% or at least about 92%,or at least about 93%, or at least about 94%, or at least about 95%, orat least about 96%, or at least about 97%, or at least about 98%, or atleast about 98.5%, or at least about 98.7%, or at least about 99%, or atleast about 99.1%, or at least about 99.2%, or at least about 99.3%, orat least about 99.4%, or at least about 99.5%, or at least about 99.6%or at least about 99.7%, or at least about 99.8%, or at least about99.9%) thereof) of pandemic virus strains. In alternative embodiments,the HA and NA polynucleotides of the invention exclude the NCR, butinclude the ORF (including fragments and variants (e.g., at least about60%, or at least 70%, or at least 80%, or at least 90%, or at leastabout 91% or at least about 92%, or at least about 93%, or at leastabout 94%, or at least about 95%, or at least about 96%, or at leastabout 97%, or at least about 98%, or at least about 98.5%, or at leastabout 98.7%, or at least about 99%, or at least about 99.1%, or at leastabout 99.2%, or at least about 99.3%, or at least about 99.4%, or atleast about 99.5%, or at least about 99.6% or at least about 99.7%, orat least about 99.8%, or at least about 99.9% thereof)) of the HA and NAsequences of pandemic virus strains (e.g., SEQ ID NOS: 1-9).

The HA and NA polynucleotides of the invention, e.g., SEQ ID NO:1through SEQ ID NO:10, and fragments thereof, are optionally used in anumber of different capacities alternative to, or in addition to, thevaccines described above. Other exemplary uses are described herein forillustrative purpose and not as limitations on the actual range of uses.Different methods of construction, purification, and characterization ofthe nucleotide sequences of the invention are also described herein. Insome embodiments, nucleic acids including one or more polynucleotidesequence of the invention are favorably used as probes for the detectionof corresponding or related nucleic acids in a variety of contexts, suchas in nucleic hybridization experiments, e.g., to find and/orcharacterize homologous influenza variants (e.g., homologues to thesequences herein, etc.) infecting other species or in differentinfluenza outbreaks, etc. The probes can be either DNA or RNA molecules,such as restriction fragments of genomic or cloned DNA, cDNAs, PCRamplification products, transcripts, and oligonucleotides, and can varyin length from oligonucleotides as short as about 10 nucleotides inlength to full length sequences or cDNAs in excess of 1 kb or more. Forexample, in some embodiments, a probe of the invention includes apolynucleotide sequence or subsequence selected, e.g., from among SEQ IDNO: 1 through SEQ ID NO: 10, or sequences complementary thereto.Alternatively, polynucleotide sequences that are variants of one of theabove-designated sequences are used as probes. Most typically, suchvariants include one or a few conservative nucleotide variations. Forexample, pairs (or sets) of oligonucleotides can be selected, in whichthe two (or more) polynucleotide sequences are conservative variationsof each other, wherein one polynucleotide sequence correspondsidentically to a first variant or and the other(s) correspondsidentically to additional variants. Such pairs of oligonucleotide probesare particularly useful, e.g., for specific hybridization experiments todetect polymorphic nucleotides or to, e.g., detect homologous influenzaHA and NA variants, e.g., homologous to the current HA and NA sequences,infecting other species or present in different (e.g., either temporallyand/or geographically different) influenza outbreaks. In otherapplications, probes are selected that are more divergent, that isprobes that are at least about 91% (or about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 98.5%, about 98.7%,about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about99.5%, or about 99.6% or more about 99.7%, about 99.8%, about 99.9% ormore) identical are selected.

The probes of the invention, e.g., as exemplified by sequences derivedfrom the sequences herein, can also be used to identify additionaluseful polynucleotide sequences according to procedures routine in theart. In one set of embodiments, one or more probes, as described above,are utilized to screen libraries of expression products or chromosomalsegments (e.g., expression libraries or genomic libraries) to identifyclones that include sequences identical to, or with significant sequencesimilarity to, e.g., one or more probe of the sequences herein, i.e.,variants, homologues, etc. It will be understood that in addition tosuch physical methods as library screening, computer assistedbioinformatic approaches, e.g., BLAST and other sequence homology searchalgorithms, and the like, can also be used for identifying relatedpolynucleotide sequences. Polynucleotide sequences identified in thismanner are also a feature of the invention.

Oligonucleotide probes are optionally produced via a variety of methodswell known to those skilled in the art. Most typically, they areproduced by well known synthetic methods, such as the solid phasephosphoramidite triester method described by Beaucage and Caruthers(1981) Tetrahedron Letts 22(20):1859-1862, e.g., using an automatedsynthesizer, or as described in Needham-Van Devanter et al. (1984) NuclAcids Res, 12:6159-6168. Oligonucleotides can also be custom made andordered from a variety of commercial sources known to persons of skill.Purification of oligonucleotides, where necessary, is typicallyperformed by either native acrylamide gel electrophoresis or byanion-exchange HPLC as described in Pearson and Regnier (1983) J Chrom255:137-149. The sequence of the synthetic oligonucleotides can beverified using the chemical degradation method of Maxam and Gilbert(1980) in Grossman and Moldave (eds.) Academic Press, New York, Methodsin Enzymology 65:499-560. Custom oligos can also easily be ordered froma variety of commercial sources known to persons of skill.

In other circumstances, e.g., relating to attributes of cells ororganisms expressing the polynucleotides and polypeptides of theinvention (e.g., those harboring virus comprising the sequences of theinvention), probes that are polypeptides, peptides or antibodies arefavorably utilized. For example, isolated or recombinant polypeptides,polypeptide fragments and peptides derived from any of the amino acidsequences of the invention (e.g., SEQ ID NO: 11-20) and/or encoded bypolynucleotide sequences of the invention, e.g., selected from SEQ IDNO: 1 through SEQ ID NO: 10, are favorably used to identify and isolateantibodies, e.g., from phage display libraries, combinatorial libraries,polyclonal sera, and the like. Polypeptide fragments of the inventionsinclude a peptide or polypeptide comprising an amino acid sequence of atleast 5 contiguous amino acid residues, or at least 10 contiguous aminoacid residues, or at least 15 contiguous amino acid residues, or atleast 20 contiguous amino acid residues, or at least 25 contiguous aminoacid residues, or at least 40 contiguous amino acid residues, or atleast 50 contiguous amino acid residues, or at least 60 contiguous aminoresidues, or at least 70 contiguous amino acid residues, or at leastcontiguous 80 amino acid residues, or at least contiguous 90 amino acidresidues, or at least contiguous 100 amino acid residues, or at leastcontiguous 125 amino acid residues, or at least 150 contiguous aminoacid residues, or at least contiguous 175 amino acid residues, or atleast contiguous 200 amino acid residues, or at least contiguous 250amino acid residues, or at least contiguous 350, or at least contiguous400, or at least contiguous 450, or at least contiguous 500, or at leastcontiguous 550 amino acid residues of the amino acid sequence an HA orNA polypeptide of the invention (e.g., SEQ ID Nos: 11-20).Polynucleotides encoding said polypeptide fragments and antibodies thatspecifically bind said polypeptides are also preferred embodiments ofthe invention.

Antibodies specific for any a polypeptide sequence or subsequence, e.g.,of SEQ ID NO: 11 through SEQ ID NO: 20, and/or encoded by polynucleotidesequences of the invention, e.g., selected from SEQ ID NO: 1 through SEQID NO: 10, are likewise valuable as probes for evaluating expressionproducts, e.g., from cells or tissues. In addition, antibodies areparticularly suitable for evaluating expression of proteins comprisingamino acid subsequences, e.g., of those given herein, or encoded bypolynucleotides sequences of the invention, e.g., selected from thoseshown herein, in situ, in a tissue array, in a cell, tissue or organism,e.g., an organism infected by an unidentified influenza virus or thelike. Antibodies can be directly labeled with a detectable reagent, ordetected indirectly by labeling of a secondary antibody specific for theheavy chain constant region (i.e., isotype) of the specific antibody.Additional details regarding production of specific antibodies areprovided below.

Diagnostic Assays

The nucleic acid sequences of the present invention can be used indiagnostic assays to detect influenza (and/or hemagglutinin and/orneuraminidase) in a sample, to detect hemagglutinin-like and/orneuraminidase-like sequences, and to detect strain differences inclinical isolates of influenza using either chemically synthesized orrecombinant polynucleotide fragments, e.g., selected from the sequencesherein. For example, fragments of the hemagglutinin and/or neuraminidasesequences comprising at least between 10 and 20 nucleotides can be usedas primers to amplify nucleic acids using polymerase chain reaction(PCR) methods well known in the art (e.g., reverse transcription-PCR)and as probes in nucleic acid hybridization assays to detect targetgenetic material such as influenza RNA in clinical specimens.

The probes of the invention, e.g., as exemplified by unique subsequencesselected from those given herein, can also be used to identifyadditional useful polynucleotide sequences (such as to characterizeadditional strains of influenza) according to procedures routine in theart. In one set of preferred embodiments, one or more probes, asdescribed above, are utilized to screen libraries of expression productsor cloned viral nucleic acids (i.e., expression libraries or genomiclibraries) to identify clones that include sequences identical to, orwith significant sequence identity to the sequences herein. In turn,each of these identified sequences can be used to make probes, includingpairs or sets of variant probes as described above. It will beunderstood that in addition to such physical methods as libraryscreening, computer assisted bioinformatic approaches, e.g., BLAST andother sequence homology search algorithms, and the like, can also beused for identifying related polynucleotide sequences.

The probes of the invention are particularly useful for detecting thepresence and for determining the identity of influenza nucleic acids incells, tissues or other biological samples (e.g., a nasal wash orbronchial lavage). For example, the probes of the invention arefavorably utilized to determine whether a biological sample, such as asubject (e.g., a human subject) or model system (such as a cultured cellsample) has been exposed to, or become infected with influenza, orparticular strain(s) of influenza. Detection of hybridization of theselected probe to nucleic acids originating in (e.g., isolated from) thebiological sample or model system is indicative of exposure to orinfection with the virus (or a related virus) from which the probepolynucleotide is selected.

It will be appreciated that probe design is influenced by the intendedapplication. For example, where several allele-specific probe-targetinteractions are to be detected in a single assay, e.g., on a single DNAchip, it is desirable to have similar melting temperatures for all ofthe probes. Accordingly, the lengths of the probes are adjusted so thatthe melting temperatures for all of the probes on the array are closelysimilar (it will be appreciated that different lengths for differentprobes may be needed to achieve a particular T_(m) where differentprobes have different GC contents). Although melting temperature is aprimary consideration in probe design, other factors are optionally usedto further adjust probe construction, such as selecting against primerself-complementarity and the like.

Vectors, Promoters and Expression Systems

The present invention includes recombinant constructs incorporating oneor more of the nucleic acid sequences described herein. Such constructsoptionally include a vector, for example, a plasmid, a cosmid, a phage,a virus, a bacterial artificial chromosome (BAC), a yeast artificialchromosome (YAC), etc., into which one or more of the polynucleotidesequences of the invention, e.g., comprising any of SEQ ID NO: 1 throughSEQ ID NO:10, or a subsequence thereof etc., has been inserted, in aforward or reverse orientation. For example, the inserted nucleic acidcan include a viral chromosomal sequence or cDNA including all or partof at least one of the polynucleotide sequences of the invention. In oneembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available.

The polynucleotides of the present invention can be included in any oneof a variety of vectors suitable for generating sense or antisense RNA,and optionally, polypeptide (or peptide) expression products (e.g., ahemagglutinin and/or neuraminidase molecule of the invention, orfragments thereof). Such vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; baculovirus; yeast plasmids; vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associatedvirus, retroviruses and many others (e.g., pCDL). Any vector that iscapable of introducing genetic material into a cell, and, if replicationis desired, which is replicable in the relevant host can be used.

In an expression vector, the HA and/or NA polynucleotide sequence ofinterest is physically arranged in proximity and orientation to anappropriate transcription control sequence (e.g., promoter, andoptionally, one or more enhancers) to direct mRNA synthesis. That is,the polynucleotide sequence of interest is operably linked to anappropriate transcription control sequence. Examples of such promotersinclude: LTR or SV40 promoter, E. coli lac or tip promoter, phage lambdaP_(L) promoter, and other promoters known to control expression of genesin prokaryotic or eukaryotic cells or their viruses.

A variety of promoters are suitable for use in expression vectors forregulating transcription of influenza virus genome segments. In certainembodiments, the cytomegalovirus (CMV) DNA dependent RNA Polymerase II(Pol II) promoter is utilized. If desired, e.g., for regulatingconditional expression, other promoters can be substituted which induceRNA transcription under the specified conditions, or in the specifiedtissues or cells. Numerous viral and mammalian, e.g., human promotersare available, or can be isolated according to the specific applicationcontemplated. For example, alternative promoters obtained from thegenomes of animal and human viruses include such promoters as theadenovirus (such as Adenovirus 2), papilloma virus, hepatitis-B virus,polyoma virus, and Simian Virus 40 (SV40), and various retroviralpromoters. Mammalian promoters include, among many others, the actinpromoter, immunoglobulin promoters, heat-shock promoters, and the like.

Transcription is optionally increased by including an enhancer sequence.Enhancers are typically short, e.g., 10-500 bp, cis-acting DNA elementsthat act in concert with a promoter to increase transcription. Manyenhancer sequences have been isolated from mammalian genes (hemoglobin,elastase, albumin, alpha-fetoprotein, and insulin), and eukaryotic cellviruses. The enhancer can be spliced into the vector at a position 5′ or3′ to the heterologous coding sequence, but is typically inserted at asite 5′ to the promoter. Typically, the promoter, and if desired,additional transcription enhancing sequences are chosen to optimizeexpression in the host cell type into which the heterologous DNA is tobe introduced (Scharf et al. (1994) Heat stress promoters andtranscription factors Results Probl Cell Differ 20:125-62; Kriegler etal. (1990) Assembly of enhancers, promoters, and splice signals tocontrol expression of transferred genes Methods in Enzymol 185: 512-27).Optionally, the amplicon can also contain a ribosome binding site or aninternal ribosome entry site (IRES) for translation initiation.

The vectors of the invention also favorably include sequences necessaryfor the termination of transcription and for stabilizing the mRNA, suchas a polyadenylation site or a terminator sequence. Such sequences arecommonly available from the 5′ and, occasionally 3′, untranslatedregions of eukaryotic or viral DNAs or cDNAs. In one embodiment, theSV40 polyadenylation signal sequences can provide a bi-directionalpolyadenylation site that insulates transcription of (+) strand mRNAmolecules from the Poll promoter initiating replication of the (−)strand viral genome.

In addition, as described above, the expression vectors optionallyinclude one or more selectable marker genes to provide a phenotypictrait for selection of transformed host cells, in addition to genespreviously listed, markers such as dihydrofolate reductase or neomycinresistance are suitable for selection in eukaryotic cell culture.

The vector containing the appropriate nucleic acid sequence as describedabove, as well as an appropriate promoter or control sequence, can beemployed to transform a host cell permitting expression of the protein.While the vectors of the invention can be replicated in bacterial cells,most frequently it will be desirable to introduce them into mammaliancells, e.g., Vero cells, BHK cells, MDCK cell, 293 cells, COS cells, orthe like, for the purpose of expression.

As described elsewhere, the HA and NA sequences herein, in variousembodiments, can be comprised within plasmids involved in plasmid-rescuereassortment. See, e.g., U.S. Application Nos. 60/420,708, filed Oct.23, 2002; 60/574,117, filed May 24, 2004; 10/423,828, filed Apr. 25,2003; 60/578,962, filed Jun. 12, 2004; and 10/870,690 filed Jun. 16,2004; and US20020164770, which are incorporated by reference herein. Forexample, preferred expression vectors of the invention include, but arenot limited to, vectors comprising pol I promoter and terminatorsequences or vectors using both the pol I and pol II promoters “thepolI/polII promoter system” (e.g., Zobel et al., Nucl. Acids Res. 1993,21:3607; US20020164770; Neumann et al., Proc. Natl. Acad. Sci. USA 1999,96:9345; Fodor et al., J. Virol. 1999, 73:9679; and US20030035814). Thereassortants produced can include the HA and NA genes arranged with the6 other influenza genes from the A/Ann Arbor/6/60 donor strain (and/orderivatives and modifications thereof), the PR8 donor strain backbone,the A/Leningrad/17 donor strain backbone, etc. Other backbone strainsare described, for example, in 20040137013 and 20030147916, which areincorporated by reference herein.

Additional Expression Elements

Most commonly, the genome segment encoding the influenza virus HA and/orNA protein includes any additional sequences necessary for itsexpression, including translation into a functional viral protein. Inother situations, a minigene, or other artificial construct encoding theviral proteins, e.g., an HA and/or NA protein, can be employed. Again,in such case, it is often desirable to include specific initiationsignals that aid in the efficient translation of the heterologous codingsequence. These signals can include, e.g., the ATG initiation codon andadjacent sequences. To insure translation of the entire insert, theinitiation codon is inserted in the correct reading frame relative tothe viral protein. Exogenous transcriptional elements and initiationcodons can be of various origins, both natural and synthetic. Theefficiency of expression can be enhanced by the inclusion of enhancersappropriate to the cell system in use.

If desired, polynucleotide sequences encoding additional expressedelements, such as signal sequences, secretion or localization sequences,and the like can be incorporated into the vector, usually, in-frame withthe polynucleotide sequence of interest, e.g., to target polypeptideexpression to a desired cellular compartment, membrane, or organelle, orto direct polypeptide secretion to the periplasmic space or into thecell culture media. Such sequences are known to those of skill, andinclude secretion leader peptides, organelle targeting sequences (e.g.,nuclear localization sequences, ER retention signals, mitochondrialtransit sequences), membrane localization/anchor sequences (e.g., stoptransfer sequences, GPI anchor sequences), and the like.

Where translation of a polypeptide encoded by a nucleic acid sequence ofthe invention is desired, additional translation specific initiationsignals can improve the efficiency of translation. These signals caninclude, e.g., an ATG initiation codon and adjacent sequences, an IRESregion, etc. In some cases, for example, full-length cDNA molecules orchromosomal segments including a coding sequence incorporating, e.g., apolynucleotide sequence of the invention (e.g., as in the sequencesherein), a translation initiation codon and associated sequence elementsare inserted into the appropriate expression vector simultaneously withthe polynucleotide sequence of interest. In such cases, additionaltranslational control signals frequently are not required. However, incases where only a polypeptide coding sequence, or a portion thereof, isinserted, exogenous translational control signals, including, e.g., anATG initiation codon is often provided for expression of the relevantsequence. The initiation codon is put in the correct reading frame toensure transcription of the polynucleotide sequence of interest.Exogenous transcriptional elements and initiation codons can be ofvarious origins, both natural and synthetic. The efficiency ofexpression can be enhanced by the inclusion of enhancers appropriate tothe cell system in use (see, e.g., Scharf D. et al. (1994) Results ProblCell Differ 20:125-62; Bittner et al. (1987) Methods in Enzymol153:516-544).

Production of Recombinant Virus

Negative strand RNA viruses can be genetically engineered and recoveredusing a recombinant reverse genetics approach (see, e.g., U.S. Pat. No.5,166,057 to Palese et al.). Such method was originally applied toengineer influenza viral genomes (Luytjes et al. (1989) Cell59:1107-1113; Enami et al. (1990) Proc. Natl. Acad. Sci. USA92:11563-11567), and has been successfully applied to a wide variety ofsegmented and nonsegmented negative strand RNA viruses, e.g., rabies(Schnell et al. (1994) EMBO J. 13: 4195-4203); VSV (Lawson et al. (1995)Proc. Natl. Acad. Sci. USA 92: 4477-4481); measles virus (Radecke et al.(1995) EMBO J. 14:5773-5784); rinderpest virus (Baron & Barrett (1997)J. Virol. 71: 1265-1271); human parainfluenza virus (Hoffman & Banerjee(1997) J. Virol. 71: 3272-3277; Dubin et al. (1997) Virology235:323-332); SV5 (He et al. (1997) Virology 237:249-260); caninedistemper virus (Gassen et al. (2000) J. Virol. 74:10737-44); and Sendaivirus (Park et al. (1991) Proc. Natl. Acad. Sci. USA 88: 5537-5541; Katoet al. (1996) Genes to Cells 1:569-579). Those of skill in the art willbe familiar with these and similar techniques to produce influenza viruscomprising the HA and NA sequences of the invention. Recombinantinfluenza viruses produced according to such methods are also a featureof the invention, as are recombinant influenza virus comprising one ormore nucleic acids and/or polypeptides of the invention.

Cell Culture and Expression Hosts

The present invention also relates to host cells that are introduced(transduced, transformed or transfected) with vectors of the invention,and the production of polypeptides of the invention by recombinanttechniques. Host cells are genetically engineered (i.e., transduced,transformed or transfected) with a vector, such as an expression vector,of this invention. As described above, the vector can be in the form ofa plasmid, a viral particle, a phage, etc. Examples of appropriateexpression hosts include: bacterial cells, such as E. coli,Streptomyces, and Salmonella typhimurium; fungal cells, such asSaccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; orinsect cells such as Drosophila and Spodoptera frugiperda.

Most commonly, mammalian cells are used to culture the HA and NAmolecules of the invention. Suitable host cells for the replication ofinfluenza virus include, e.g., Vero cells, BHK cells, MDCK cells, 293cells and COS cells, including 293T cells, COS7 cells or the like.Commonly, co-cultures including two of the above cell lines, e.g., MDCKcells and either 293T or COS cells are employed at a ratio, e.g., of1:1, to improve replication efficiency. Typically, cells are cultured ina standard commercial culture medium, such as Dulbecco's modifiedEagle's medium supplemented with serum (e.g., 10% fetal bovine serum),or in serum free medium, under controlled humidity and CO₂ concentrationsuitable for maintaining neutral buffered pH (e.g., at pH between 7.0and 7.2). Optionally, the medium contains antibiotics to preventbacterial growth, e.g., penicillin, streptomycin, etc., and/oradditional nutrients, such as L-glutamine, sodium pyruvate,non-essential amino acids, additional supplements to promote favorablegrowth characteristics, e.g., trypsin, β-mercaptoethanol, and the like.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants, or amplifying the inserted polynucleotide sequences. Theculture conditions, such as temperature, pH and the like, are typicallythose previously used with the particular host cell selected forexpression, and will be apparent to those skilled in the art and in thereferences cited herein, including, e.g., Freshney (1994) Culture ofAnimal Cells, a Manual of Basic Technique, 3^(rd) edition, Wiley-Liss,New York and the references cited therein. Other helpful referencesinclude, e.g., Paul (1975) Cell and Tissue Culture, 5^(th) ed.,Livingston, Edinburgh; Adams (1980) Laboratory Techniques inBiochemistry and Molecular Biology-Cell Culture for Biochemists, Workand Burdon (eds.) Elsevier, Amsterdam. Additional details regardingtissue culture procedures of particular interest in the production ofinfluenza virus in vitro include, e.g., Merten et al. (1996) Productionof influenza virus in cell cultures for vaccine preparation. in Cohenand Shafferman (eds.) Novel Strategies in Design and Production ofVaccines, which is incorporated herein in its entirety for all purposes.Additionally, variations in such procedures adapted to the presentinvention are readily determined through routine experimentation andwill be familiar to those skilled in the art.

Cells for production of influenza virus (e.g., having the HA and/or NAsequences of the invention) can be cultured in serum-containing or serumfree medium. In some case, e.g., for the preparation of purifiedviruses, it is typically desirable to grow the host cells in serum freeconditions. Cells can be cultured in small scale, e.g., less than 25 mlmedium, culture tubes or flasks or in large flasks with agitation, inrotator bottles, or on microcarrier beads (e.g., DEAE-Dextranmicrocarrier beads, such as Dormacell, Pfeifer & Langen; Superbead, FlowLaboratories; styrene copolymer-tri-methylamine beads, such as Hillex,SoloHill, Ann Arbor) in flasks, bottles or reactor cultures.Microcarrier beads are small spheres (in the range of 100-200 microns indiameter) that provide a large surface area for adherent cell growth pervolume of cell culture. For example a single liter of medium can includemore than 20 million microcarrier beads providing greater than 8000square centimeters of growth surface. For commercial production ofviruses, e.g., for vaccine production, it is often desirable to culturethe cells in a bioreactor or fermenter. Bioreactors are available involumes from under 1 liter to in excess of 100 liters, e.g., Cyto3Bioreactor (Osmonics, Minnetonka, Minn.); NBS bioreactors (New BrunswickScientific, Edison, N.J.); laboratory and commercial scale bioreactorsfrom B. Braun Biotech International (B. Braun Biotech, Melsungen,Germany).

Regardless of the culture volume, in many desired aspects of the currentinvention, it is important that the cultures be maintained at anappropriate temperature, to insure efficient recovery of recombinantand/or reassortant influenza virus using temperature dependent multiplasmid systems (see, e.g., Multi-Plasmid System for the Production ofInfluenza Virus, U.S. Application No. 60/420,708, filed Oct. 23, 2002,U.S. application Ser. No. 10/423,828, filed Apr. 25, 2003, and U.S.Application No. 60/574,117, filed May 24, 2004), heating of virussolutions for filtration, etc. Typically, a regulator, e.g., athermostat, or other device for sensing and maintaining the temperatureof the cell culture system and/or other solution, is employed to insurethat the temperature is at the correct level during the appropriateperiod (e.g., virus replication, etc.).

In some embodiments herein (e.g., wherein reassorted viruses are to beproduced from segments on vectors) vectors comprising influenza genomesegments are introduced (e.g., transfected) into host cells according tomethods well known in the art for introducing heterologous nucleic acidsinto eukaryotic cells, including, e.g., calcium phosphateco-precipitation, electroporation, microinjection, lipofection, andtransfection employing polyamine transfection reagents. For example,vectors, e.g., plasmids, can be transfected into host cells, such as COScells, 293T cells or combinations of COS or 293T cells and MDCK cells,using the polyamine transfection reagent TranslT-LT1 (Mirus) accordingto the manufacturer's instructions in order to produce reassortedviruses, etc. Thus, in one example, approximately 1 μg of each vector isintroduced into a population of host cells with approximately 2 μl ofTransIT-LT1 diluted in 160 μl medium, preferably serum-free medium, in atotal volume of 200 μl. The DNA:transfection reagent mixtures areincubated at room temperature for 45 minutes followed by addition of 800μl of medium. The transfection mixture is added to the host cells, andthe cells are cultured as described via other methods well known tothose skilled in the art. Accordingly, for the production of recombinantor reassortant viruses in cell culture, vectors incorporating each ofthe 8 genome segments, (PB2, FBI, PA, NP, M, NS, HA and NA, e.g., of theinvention) are mixed with approximately 20 μl TranslT-LT1 andtransfected into host cells. Optionally, serum-containing medium isreplaced prior to transfection with serum-free medium, e.g., Opti-MEM I,and incubated for 4-6 hours.

Alternatively, electroporation can be employed to introduce such vectorsincorporating influenza genome segments into host cells. For example,plasmid vectors incorporating an influenza A or influenza B virus arefavorably introduced into Vero cells using electroporation according tothe following procedure. In brief, approximately 5×10⁶ Vero cells, e.g.,grown in Modified Eagle's Medium (MEM) supplemented with 10% FetalBovine Serum (FBS) are resuspended in 0.4 ml OptiMEM and placed in anelectroporation cuvette. Twenty micrograms of DNA in a volume of up to25 μl is added to the cells in the cuvette, which is then mixed gentlyby tapping. Electroporation is performed according to the manufacturer'sinstructions (e.g., BioRad Gene Pulser II with Capacitance Extender Plusconnected) at 300 volts, 950 microFarads with a time constant of between28-33 msec. The cells are remixed by gently tapping and approximately1-2 minutes following electroporation 0.7 ml MEM with 10% FBS is addeddirectly to the cuvette. The cells are then transferred to two wells ofa standard 6 well tissue culture dish containing 2 ml MEM, 10% FBS. Thecuvette is washed to recover any remaining cells and the wash suspensionis divided between the two wells. Final volume is approximately 3.5 mL.The cells are then incubated under conditions permissive for viralgrowth, e.g., at approximately 33° C. for cold adapted strains.

In mammalian host cells, a number of expression systems, such asviral-based systems, can be utilized. In cases where an adenovirus isused as an expression vector, a coding sequence is optionally ligatedinto an adenovirus transcription/translation complex consisting of thelate promoter and tripartite leader sequence. Insertion in anonessential E1 or E3 region of the viral genome will result in a viablevirus capable of expressing the polypeptides of interest in infectedhost cells (Logan and Shenk (1984) Proc Natl Acad Sci 81:3655-3659). Inaddition, transcription enhancers, such as the rous sarcoma virus (RSV)enhancer, can be used to increase expression in mammalian host cells.

A host cell strain is optionally chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed proteinin the desired fashion. Such modifications of the protein include, butare not limited to, acetylation, carboxylation, glycosylation,phosphorylation, lipidation and acylation. Post-translationalprocessing, which cleaves a precursor form into a mature form, of theprotein is sometimes important for correct insertion, folding and/orfunction. Additionally proper location within a host cell (e.g., on thecell surface) is also important. Different host cells such as COS, CHO,BHK, MDCK, 293, 293T, COS7, etc. have specific cellular machinery andcharacteristic mechanisms for such post-translational activities and canbe chosen to ensure the correct modification and processing of thecurrent introduced, foreign protein.

For long-term, high-yield production of recombinant proteins encoded by,or having subsequences encoded by, the polynucleotides of the invention,stable expression systems are optionally used. For example, cell lines,stably expressing a polypeptide of the invention, are transfected usingexpression vectors that contain viral origins of replication orendogenous expression elements and a selectable marker gene. Forexample, following the introduction of the vector, cells are allowed togrow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells that successfully express the introduced sequences. Thus,resistant clumps of stably transformed cells, e.g., derived from singlecell type, can be proliferated using tissue culture techniquesappropriate to the cell type.

Host cells transformed with a nucleotide sequence encoding a polypeptideof the invention are optionally cultured under conditions suitable forthe expression and recovery of the encoded protein from cell culture.The cells expressing said protein can be sorted, isolated and/orpurified. The protein or fragment thereof produced by a recombinant cellcan be secreted, membrane-bound, or retained intracellularly, dependingon the sequence (e.g., depending upon fusion proteins encoding amembrane retention signal or the like) and/or the vector used.

Expression products corresponding to the nucleic acids of the inventioncan also be produced in non-animal cells such as plants, yeast, fungi,bacteria and the like. In addition to Sambrook, Berger and Ausubel, allinfra, details regarding cell culture can be found in Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.)The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.

In bacterial systems, a number of expression vectors can be selecteddepending upon the use intended for the expressed product. For example,when large quantities of a polypeptide or fragments thereof are neededfor the production of antibodies, vectors that direct high-levelexpression of fusion proteins that are readily purified are favorablyemployed. Such vectors include, but are not limited to, multifunctionalE. coli cloning and expression vectors such as BLUESCRIPT (Stratagene),in which the coding sequence of interest, e.g., sequences comprisingthose found herein, etc., can be ligated into the vector in-frame withsequences for the amino-terminal translation initiating methionine andthe subsequent 7 residues of beta-galactosidase producing acatalytically active beta galactosidase fusion protein; pIN vectors (VanHeeke & Schuster (1989) J Biol Chem 264:5503-5509); pET vectors(Novagen, Madison Wis.); and the like. Similarly, in the yeastSaccharomyces cerevisiae a number of vectors containing constitutive orinducible promoters such as alpha factor, alcohol oxidase and PGH can beused for production of the desired expression products. For reviews, seeAusubel, infra, and Grant et al., (1987); Methods in Enzymology153:516-544.

Nucleic Acid Hybridization

Comparative hybridization can be used to identify nucleic acids (e.g.,SEQ ID NO: 1-10) of the invention, including conservative variations ofnucleic acids of the invention. This comparative hybridization method isa preferred method of distinguishing nucleic acids of the invention. Inaddition, target nucleic acids which hybridize to the nucleic acidsrepresented by, e.g., those shown herein under high, ultra-high andultra-ultra-high stringency conditions are features of the invention.Examples of such nucleic acids include those with one or a few silent orconservative nucleic acid substitutions as compared to a given nucleicacid sequence.

A test target nucleic acid is said to specifically hybridize to a probenucleic acid when it hybridizes at least one-half as well to the probeas to the perfectly matched complementary target, i.e., with a signal tonoise ratio at least one-half as high as hybridization of the probe andtarget under conditions in which a perfectly matched probe binds to aperfectly matched complementary target with a signal to noise ratio thatis at least about 5×-10× as high as that observed for hybridization toany of the unmatched target nucleic acids.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well-characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. Numerous protocols for nucleic acidhybridization are well known in the art. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes part I chapter 2, “Overview of principles ofhybridization and the strategy of nucleic acid probe assays,” (Elsevier,N.Y.), as well as in Ausubel, Sambrook, and Berger and Kimmel, allbelow. Hames and Higgins (1995) Gene Probes 1 IRL Press at OxfordUniversity Press, Oxford, England, (Hames and Higgins 1) and Hames andHiggins (1995) Gene Probes 2 IRL Press at Oxford University Press,Oxford, England (Hames and Higgins 2) provide details on the synthesis,labeling, detection and quantification of DNA and RNA, includingoligonucleotides.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of stringent wash conditions comprises a 0.2×SSCwash at 65° C. for 15 minutes (see, Sambrook, infra for a description ofSSC buffer and other nucleic acid hybridization parameters). Often thehigh stringency wash is preceded by a low stringency wash to removebackground probe signal. An example low stringency wash is 2×SSC at 40°C. for 15 minutes. In general, a signal to noise ratio of 5× (or higher)than that observed for an unrelated probe in the particularhybridization assay indicates detection of a specific hybridization.

After hybridization, unhybridized nucleic acids can be removed by aseries of washes, the stringency of which can be adjusted depending uponthe desired results. Low stringency washing conditions (e.g., usinghigher salt and lower temperature) increase sensitivity, but can producenonspecific hybridization signals and high background signals. Higherstringency conditions (e.g., using lower salt and higher temperaturethat is closer to the T_(m)) lower the background signal, typically withprimarily the specific signal remaining. See, also, Rapley, R. andWalker, J. M. eds., Molecular Biomethods Handbook (Humana Press, Inc.1998).

“Stringent hybridization wash conditions” in the context of nucleic acidhybridization experiments such as Southern and northern hybridizationsare sequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993), supra, and in Hames and Higgins, 1 and 2.Stringent hybridization and wash conditions can easily be determinedempirically for any test nucleic acid. For example, in determininghighly stringent hybridization and wash conditions, the hybridizationand wash conditions are gradually increased (e.g., by increasingtemperature, decreasing salt concentration, increasing detergentconcentration and/or increasing the concentration of organic solventssuch as formalin in the hybridization or wash), until a selected set ofcriteria is met. For example, the hybridization and wash conditions aregradually increased until a probe binds to a perfectly matchedcomplementary target with a signal to noise ratio that is at least 5× ashigh as that observed for hybridization of the probe to an unmatchedtarget.

In general, a signal to noise ratio of at least 2× (or higher, e.g., atleast 5×, 10×, 20×, 50×, 100×, or more) than that observed for anunrelated probe in the particular hybridization assay indicatesdetection of a specific hybridization. Detection of at least stringenthybridization between two sequences in the context of the presentinvention indicates relatively strong structural similarity to, e.g.,the nucleic acids of the present invention provided in the sequencelistings herein.

“Very stringent” conditions are selected to be equal to the thermalmelting point (T_(m)) for a particular probe. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetest sequence hybridizes to a perfectly matched probe. For the purposesof the present invention, generally, “highly stringent” hybridizationand wash conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence at a defined ionic strength and pH (as notedbelow, highly stringent conditions can also be referred to incomparative terms). Target sequences that are closely related oridentical to the nucleotide sequence of interest (e.g., “probe”) can beidentified under stringent or highly stringent conditions. Lowerstringency conditions are appropriate for sequences that are lesscomplementary.

“Ultra high-stringency” hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of a probe to a perfectlymatched complementary target nucleic acid is at least 10× as high asthat observed for hybridization to any unmatched target nucleic acids. Atarget nucleic acid which hybridizes to a probe under such conditions,with a signal to noise ratio of at least one-half that of the perfectlymatched complementary target nucleic acid is said to bind to the probeunder ultra-high stringency conditions.

In determining stringent or highly stringent hybridization (or even morestringent hybridization) and wash conditions, the hybridization and washconditions are gradually increased (e.g., by increasing temperature,decreasing salt concentration, increasing detergent concentration and/orincreasing the concentration of organic solvents, such as formamide, inthe hybridization or wash), until a selected set of criteria are met.For example, the hybridization and wash conditions are graduallyincreased until a probe comprising one or more polynucleotide sequencesof the invention, e.g., sequences or unique subsequences selected fromthose given herein (e.g., SEQ ID NO: 1-10) and/or complementarypolynucleotide sequences, binds to a perfectly matched complementarytarget (again, a nucleic acid comprising one or more nucleic acidsequences or subsequences selected from those given herein and/orcomplementary polynucleotide sequences thereof), with a signal to noiseratio that is at least 2× (and optionally 5×, 10×, or 100× or more) ashigh as that observed for hybridization of the probe to an unmatchedtarget (e.g., a polynucleotide sequence comprising one or more sequencesor subsequences selected from known influenza sequences present inpublic databases such as GenBank at the time of filing, and/orcomplementary polynucleotide sequences thereof), as desired.

Using the polynucleotides of the invention, or subsequences thereof,novel target nucleic acids can be obtained; such target nucleic acidsare also a feature of the invention. For example, such target nucleicacids include sequences that hybridize under stringent conditions to aunique oligonucleotide probe corresponding to any of the polynucleotidesof the invention, e.g., SEQ ID NO: 1-10).

Similarly, even higher levels of stringency can be determined bygradually increasing the hybridization and/or wash conditions of therelevant hybridization assay. For example, those in which the stringencyof hybridization and wash conditions are increased until the signal tonoise ratio for binding of the probe to the perfectly matchedcomplementary target nucleic acid is at least 10×, 20×, 50×, 100×, or500× or more as high as that observed for hybridization to any unmatchedtarget nucleic acids. The particular signal will depend on the labelused in the relevant assay, e.g., a fluorescent label, a colorimetriclabel, a radioactive label, or the like. A target nucleic acid whichhybridizes to a probe under such conditions, with a signal to noiseratio of at least one-half that of the perfectly matched complementarytarget nucleic acid is said to bind to the probe under ultra-ultra-highstringency conditions and are also features of the invention.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Cloning, Mutagenesis and Expression of Biomolecules of Interest

General texts which describe molecular biological techniques, which areapplicable to the present invention, such as cloning, mutation, cellculture and the like, include Berger and Kimmel, Guide to MolecularCloning Techniques, Methods in Enzymology volume 152 Academic Press,Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—ALaboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 2000 (“Sambrook”) and Current Protocols inMolecular Biology, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley& Sons, Inc., (supplemented through 2002) (“Ausubel”)). These textsdescribe mutagenesis, the use of vectors, promoters and many otherrelevant topics related to, e.g., the generation of HA and/or NAmolecules, etc.

Various types of mutagenesis are optionally used in the presentinvention, e.g., to produce and/or isolate, e.g., novel or newlyisolated HA and/or NA molecules and/or to further modify/mutate thepolypeptides (e.g., HA and NA molecules as in SEQ ID NO: 11-20) of theinvention. They include but are not limited to site-directed, randompoint mutagenesis, homologous recombination (DNA shuffling), mutagenesisusing uracil containing templates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA or the like. Additional suitable methods include pointmismatch repair, mutagenesis using repair-deficient host strains,restriction-selection and restriction-purification, deletionmutagenesis, mutagenesis by total gene synthesis, double-strand breakrepair, and the like. Mutagenesis, e.g., involving chimeric constructs,is also included in the present invention. In one embodiment,mutagenesis can be guided by known information of the naturallyoccurring molecule or altered or mutated naturally occurring molecule,e.g., sequence, sequence comparisons, physical properties, crystalstructure or the like.

The above texts and examples found herein describe these procedures aswell as the following publications (and references cited within):Sieber, et al., Nature Biotechnology, 19:456-460 (2001); Ling et al.,Approaches to DNA mutagenesis: an overview, Anal Biochem 254(2): 157-178(1997); Dale et al., Oligonucleotide-directed random mutagenesis usingthe phosphorothioate method, Methods Mol Biol 57:369-374 (1996); I. A.Lorimer, I. Pastan, Nucleic Acids Res 23, 3067-8 (1995); W. P. C.Stemmer, Nature 370, 389-91 (1994); Arnold, Protein engineering forunusual environments, Current Opinion in Biotechnology 4:450-455 (1993);Bass et al., Mutant Trp repressors with new DNA-binding specificities,Science 242:240-245 (1988); Fritz et al., Oligonucleotide-directedconstruction of mutations: a gapped duplex DNA procedure withoutenzymatic reactions in vitro, Nucl Acids Res 16: 6987-6999 (1988);Kramer et al., Improved enzymatic in vitro reactions in the gappedduplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl Acids Res 16: 7207 (1988); Sakamar and Khorana, Totalsynthesis and expression of a gene for the a-subunit of bovine rod outersegment guanine nucleotide-binding protein (transducin), Nucl Acids Res14: 6361-6372 (1988); Sayers et al., Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis, Nucl AcidsRes 16:791-802 (1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl Acids Res16: 803-814; Carter, Improved oligonucleotide-directed mutagenesis usingM13 vectors, Methods in Enzymol 154: 382-403 (1987); Kramer & FritzOligonucleotide-directed construction of mutations via gapped duplexDNA, Methods in Enzymol 154:350-367 (1987); Kunkel, The efficiency ofoligonucleotide directed mutagenesis, in Nucleic Acids & MolecularBiology (Eckstein, F. and Lilley, D. M. J. eds., Springer Verlag,Berlin)) (1987); Kunkel et al., Rapid and efficient site-specificmutagenesis without phenotypic selection, Methods in Enzymol 154,367-382 (1987); Zoller & Smith, Oligonucleotide-directed mutagenesis: asimple method using two oligonucleotide primers and a single-strandedDNA template, Methods in Enzymol 154:329-350 (1987); Carter,Site-directed mutagenesis, Biochem J 237:1-7 (1986); Eghtedarzadeh &Henikoff, Use of oligonucleotides to generate large deletions, NuclAcids Res 14: 5115 (1986); Mandecki, Oligonucleotide-directeddouble-strand break repair in plasmids of Escherichia coli: a method forsite-specific mutagenesis, Proc Natl Acad Sci USA, 83:7177-7181 (1986);Nakamaye & Eckstein, Inhibition of restriction endonuclease Nci Icleavage by phosphorothioate groups and its application tooligonucleotide-directed mutagenesis, Nucl Acids Res 14: 9679-9698(1986); Wells et al., Importance of hydrogen-bond formation instabilizing the transition state of subtilisin, Phil Trans R Soc Lond A317: 415-423 (1986); Botstein & Shortle, Strategies and applications ofin vitro mutagenesis, Science 229:1193-1201 (1985); Carter et al.,Improved oligonucleotide site-directed mutagenesis using M13 vectors,Nucl Acids Res 13: 4431-4443 (1985); Grundström et al.,Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’ genesynthesis, Nucl Acids Res 13: 3305-3316 (1985); Kunkel, Rapid andefficient site-specific mutagenesis without phenotypic selection, ProcNatl Acad Sci USA 82:488-492 (1985); Smith, In vitro mutagenesis, AnnRev Genet. 19:423-462 (1985); Taylor et al., The use ofphosphorothioate-modified DNA in restriction enzyme reactions to preparenicked DNA, Nucl Acids Res 13: 8749-8764 (1985); Taylor et al., Therapid generation of oligonucleotide-directed mutations at high frequencyusing phosphorothioate-modified DNA, Nucl Acids Res 13: 8765-8787(1985); Wells et al., Cassette mutagenesis: an efficient method forgeneration of multiple mutations at defined sites, Gene 34:315-323(1985); Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl Acids Res 12:9441-9456 (1984); Kramer et al., Point Mismatch Repair, Cell 38:879-887(1984); Nambiar et al., Total synthesis and cloning of a gene coding forthe ribonuclease S protein, Science 223: 1299-1301 (1984); Zoller &Smith, Oligonucleotide-directed mutagenesis of DNA fragments cloned intoM13 vectors, Methods in Enzymol 100:468-500 (1983); and Zoller & Smith,Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment, Nucl Acids Res 10:6487-6500 (1982). Additional detailson many of the above methods can be found in Methods in Enzymol Volume154, which also describes useful controls for trouble-shooting problemswith various mutagenesis, gene isolation, expression, and other methods.

Oligonucleotides, e.g., for use in mutagenesis of the present invention,e.g., mutating libraries of the HA and/or NA molecules of the invention,or altering such, are typically synthesized chemically according to thesolid phase phosphoramidite triester method described by Beaucage andCaruthers, Tetrahedron Letts 22(20):1859-1862, (1981) e.g., using anautomated synthesizer, as described in Needham-VanDevanter et al.,Nucleic Acids Res, 12:6159-6168 (1984).

In addition, essentially any nucleic acid can be custom or standardordered from any of a variety of commercial sources, such as The MidlandCertified Reagent Company (mcrc@oligos.com), The Great American GeneCompany (www.genco.com), ExpressGen Inc. (www.expressgen.com), OperonTechnologies Inc. (Alameda, Calif.) and many others. Similarly, peptidesand antibodies can be custom ordered from any of a variety of sources,such as PeptidoGenic (available at pkim@ccnet.com), HTI Bio-products,Inc. (www.htibio.com), BMA Biomedicals Ltd. (U.K.), Bio.Synthesis, Inc.,and many others.

The present invention also relates to host cells and organismscomprising a HA and/or NA molecule or other polypeptide and/or nucleicacid of the invention, e.g., SEQ ID NO:1-20. Host cells are geneticallyengineered (e.g., transformed, transduced or transfected) with thevectors of this invention, which can be, for example, a cloning vectoror an expression vector. The vector can be, for example, in the form ofa plasmid, a bacterium, a virus, a naked polynucleotide, or a conjugatedpolynucleotide. The vectors are introduced into cells and/ormicroorganisms by standard methods including electroporation (see, Fromet al., Proc Natl Acad Sci USA 82, 5824 (1985), infection by viralvectors, high velocity ballistic penetration by small particles with thenucleic acid either within the matrix of small beads or particles, or onthe surface (Klein et al., Nature 327, 70-73 (1987)). Berger, Sambrook,and Ausubel provide a variety of appropriate transformation methods.See, above.

Several well-known methods of introducing target nucleic acids intobacterial cells are available, any of which can be used in the presentinvention. These include: fusion of the recipient cells with bacterialprotoplasts containing the DNA, electroporation, projectile bombardment,and infection with viral vectors, etc. Bacterial cells can be used toamplify the number of plasmids containing DNA constructs of thisinvention. The bacteria are grown to log phase and the plasmids withinthe bacteria can be isolated by a variety of methods known in the art(see, for instance, Sambrook). In addition, a plethora of kits arecommercially available for the purification of plasmids from bacteria,(see, e.g., EasyPrep™, FlexiPrep™, both from Pharmacia Biotech;StrataClean™, from Stratagene; and, QIAprep™ from Qiagen). The isolatedand purified plasmids are then further manipulated to produce otherplasmids, used to transfect cells or incorporated into related vectorsto infect organisms. Typical vectors contain transcription andtranslation terminators, transcription and translation initiationsequences, and promoters useful for regulation of the expression of theparticular target nucleic acid. The vectors optionally comprise genericexpression cassettes containing at least one independent terminatorsequence, sequences permitting replication of the cassette ineukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) andselection markers for both prokaryotic and eukaryotic systems. Vectorsare suitable for replication and integration in prokaryotes, eukaryotes,or optionally both. See, Giliman & Smith, Gene 8:81 (1979); Roberts, etal., Nature, 328:731 (1987); Schneider, B., et al., Protein Expr Purif6435:10 (1995); Ausubel, Sambrook, Berger (all supra). A catalogue ofBacteria and Bacteriophages useful for cloning is provided, e.g., by theATCC, e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1992)Gherna et al. (eds.) published by the ATCC. Additional basic proceduresfor sequencing, cloning and other aspects of molecular biology andunderlying theoretical considerations are also found in Watson et al.(1992) Recombinant DNA Second Edition Scientific American Books, NY.See, above. Further vectors useful with the sequences herein areillustrated above in the section concerning production of influenzavirus for vaccines and the references cited therein.

Polypeptide Production and Recovery

Following transduction of a suitable host cell line or strain and growthof the host cells to an appropriate cell density, the selected promoteris induced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. In someembodiments, a secreted polypeptide product, e.g., a HA and/or NApolypeptide as in a secreted fusion protein form, etc., is thenrecovered from the culture medium. In other embodiments, a virusparticle containing a HA and/or a NA polypeptide of the invention isproduced from the cell. Alternatively, cells can be harvested bycentrifugation, disrupted by physical or chemical means, and theresulting crude extract retained for further purification. Eukaryotic ormicrobial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, or other methods,which are well know to those skilled in the art. Additionally, cellsexpressing a HA and/or a NA polypeptide product of the invention can beutilized without separating the polypeptide from the cell. In suchsituations, the polypeptide of the invention is optionally expressed onthe cell surface and is examined thus (e.g., by having HA and/or NAmolecules (or fragments thereof, e.g., comprising fusion proteins or thelike) on the cell surface bind antibodies, etc. Such cells are alsofeatures of the invention.

Expressed polypeptides can be recovered and purified from recombinantcell cultures by any of a number of methods well known in the art,including ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography (e.g., using any of the tagging systems known to thoseskilled in the art), hydroxylapatite chromatography, and lectinchromatography. Protein refolding steps can be used, as desired, incompleting configuration of the mature protein. Also, high performanceliquid chromatography (HPLC) can be employed in the final purificationsteps. In addition to the references noted herein, a variety ofpurification methods are well known in the art, including, e.g., thoseset forth in Sandana (1997) Bioseparation of Proteins, Academic Press,Inc.; and Bollag et al. (1996) Protein Methods, 2^(nd) EditionWiley-Liss, NY; Walker (1996) The Protein Protocols Handbook HumanaPress, NJ, Harris and Angal (1990) Protein Purification Applications: APractical Approach IRL Press at Oxford, Oxford, England; Harris andAngal Protein Purification Methods: A Practical Approach IRL Press atOxford, Oxford, England; Scopes (1993) Protein Purification: Principlesand Practice 3^(rd) Edition Springer Verlag, NY; Janson and Ryden (1998)Protein Purification: Principles, High Resolution Methods andApplications, Second Edition Wiley-VCH, NY; and Walker (1998) ProteinProtocols on CD-ROM Humana Press, NJ.

When the expressed polypeptides of the invention are produced inviruses, the viruses are typically recovered from the culture medium, inwhich infected (transfected) cells have been grown. Typically, crudemedium is clarified prior to concentration of influenza viruses. Commonmethods include ultrafiltration, adsorption on barium sulfate andelution, and centrifugation. For example, crude medium from infectedcultures can first be clarified by centrifugation at, e.g., 1000-2000×gfor a time sufficient to remove cell debris and other large particulatematter, e.g., between 10 and 30 minutes. Optionally, the clarifiedmedium supernatant is then centrifuged to pellet the influenza viruses,e.g., at 15,000×g, for approximately 3-5 hours. Following resuspensionof the virus pellet in an appropriate buffer, such as STE (0.01 MTris-HCl; 0.15 M NaCl; 0.0001 M EDTA) or phosphate buffered saline (PBS)at pH 7.4, the virus is concentrated by density gradient centrifugationon sucrose (60%-12%) or potassium tartrate (50%-10%). Either continuousor step gradients, e.g., a sucrose gradient between 12% and 60% in four12% steps, are suitable. The gradients are centrifuged at a speed, andfor a time, sufficient for the viruses to concentrate into a visibleband for recovery. Alternatively, and for most large-scale commercialapplications, virus is elutriated from density gradients using azonal-centrifuge rotor operating in continuous mode. Additional detailssufficient to guide one of skill through the preparation of influenzaviruses from tissue culture are provided, e.g., in Furminger. VaccineProduction, in Nicholson et al. (eds.) Textbook of Influenza pp.324-332; Merten et al. (1996) Production of influenza virus in cellcultures for vaccine preparation, in Cohen & Shafferman (eds.) NovelStrategies in Design and Production of Vaccines pp. 141-151, and U.S.Pat. No. 5,690,937. If desired, the recovered viruses can be stored at−80° C. in the presence of sucrose-phosphate-glutamate (SPG) as astabilizer

Alternatively, cell-free transcription/translation systems can beemployed to produce polypeptides comprising an amino acid sequence orsubsequence of, e.g., the sequences given herein such as SEQ ID NO:11-20, or encoded by the polynucleotide sequences of the invention,e.g., SEQ ID NO: 1-10. A number of suitable in vitro transcription andtranslation systems are commercially available. A general guide to invitro transcription and translation protocols is found in Tymms (1995)In vitro Transcription and Translation Protocols: Methods in MolecularBiology Volume 37, Garland Publishing, NY.

In addition, the polypeptides, or subsequences thereof, e.g.,subsequences comprising antigenic peptides, can be produced manually orby using an automated system, by direct peptide synthesis usingsolid-phase techniques (see, Stewart et al. (1969) Solid-Phase PeptideSynthesis, WH Freeman Co, San Francisco; Merrifield J (1963) J Am ChemSoc 85:2149-2154). Exemplary automated systems include the AppliedBiosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.).If desired, subsequences can be chemically synthesized separately, andcombined using chemical methods to provide full-length polypeptides.

Modified Amino Acids

Expressed polypeptides of the invention can contain one or more modifiedamino acids. The presence of modified amino acids can be advantageousin, for example, (a) increasing polypeptide serum half-life, (b)reducing/increasing polypeptide antigenicity, (c) increasing polypeptidestorage stability, etc Amino acid(s) are modified, for example,co-translationally or post-translationally during recombinant production(e.g., N-linked glycosylation at N—X—S/T motifs during expression inmammalian cells) or modified by synthetic means (e.g., via PEGylation).

Non-limiting examples of a modified amino acid include a glycosylatedamino acid, a sulfated amino acid, a prenlyated (e.g., farnesylated,geranylgeranylated) amino acid, an acetylated amino acid, an acylatedamino acid, a PEG-ylated amino acid, a biotinylated amino acid, acarboxylated amino acid, a phosphorylated amino acid, and the like, aswell as amino acids modified by conjugation to, e.g., lipid moieties orother organic derivatizing agents. References adequate to guide one ofskill in the modification of amino acids are replete throughout theliterature. Example protocols are found in Walker (1998) ProteinProtocols on CD-ROM Human Press, Towata, N.J.

Fusion Proteins

The present invention also provides fusion proteins comprising fusionsof the sequences of the invention (e.g., encoding HA and/or NApolypeptides as exampled by SEQ ID NO: 11-20) or fragments thereof with,e.g., immunoglobulins (or portions thereof), sequences encoding, e.g.,GFP (green fluorescent protein), or other similar markers, etc.Nucleotide sequences encoding such fusion proteins are another aspect ofthe invention. Fusion proteins of the invention are optionally used for,e.g., similar applications (including, e.g., therapeutic, prophylactic,diagnostic, experimental, etc. applications as described herein) as thenon-fusion proteins of the invention. In addition to fusion withimmunoglobulin sequences and marker sequences, the proteins of theinvention are also optionally fused with, e.g., sequences which allowsorting of the fusion proteins and/or targeting of the fusion proteinsto specific cell types, regions, etc.

Antibodies

The polypeptides of the invention can be used to produce antibodiesspecific for the polypeptides given herein and/or polypeptides encodedby the polynucleotides of the invention, e.g., those shown herein, andconservative variants thereof. Antibodies specific for the abovementioned polypeptides are useful, e.g., for diagnostic and therapeuticpurposes, e.g., related to the activity, distribution, and expression oftarget polypeptides.

Antibodies specific for the polypeptides of the invention can begenerated by methods well known in the art. Such antibodies can include,but are not limited to, polyclonal, monoclonal, chimeric, humanized,single chain, Fab fragments and fragments produced by an Fab expressionlibrary.

Polypeptides do not require biological activity for antibody production(e.g., full length functional hemagglutinin or neuraminidase is notrequired). However, the polypeptide or oligopeptide must be antigenic.Peptides used to induce specific antibodies typically have an amino acidsequence of at least about 4 amino acids, and often at least 5 or 10amino acids. Short stretches of a polypeptide can be fused with anotherprotein, such as keyhole limpet hemocyanin, and antibody producedagainst the chimeric molecule.

Numerous methods for producing polyclonal and monoclonal antibodies areknown to those of skill in the art, and can be adapted to produceantibodies specific for the polypeptides of the invention, and/orencoded by the polynucleotide sequences of the invention, etc. See,e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY;Paul (ed.) (1998) Fundamental Immunology, Fourth Edition,Lippincott-Raven, Lippincott Williams & Wilkins; Harlow and Lane (1989)Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY; Stites etal. (eds.) Basic and Clinical Immunology (4th ed.) Lange MedicalPublications, Los Altos, Calif., and references cited therein; Goding(1986) Monoclonal Antibodies: Principles and Practice (2d ed.) AcademicPress, New York, N.Y.; and Kohler and Milstein (1975) Nature 256:495-497. Other suitable techniques for antibody preparation includeselection of libraries of recombinant antibodies in phage or similarvectors. See, Huse et al. (1989) Science 246: 1275-1281; and Ward, etal. (1989) Nature 341: 544-546. Specific monoclonal and polyclonalantibodies and antisera will usually bind with a K_(D) of, e.g., atleast about 0.1 μM, at least about 0.01 μM or better, and, typically andat least about 0.001 μM or better.

For certain therapeutic applications, humanized antibodies aredesirable. Detailed methods for preparation of chimeric (humanized)antibodies can be found in U.S. Pat. No. 5,482,856. Additional detailson humanization and other antibody production and engineering techniquescan be found in Borrebaeck (ed.) (1995) Antibody Engineering, 2^(nd)Edition Freeman and Company, NY (Borrebaeck); McCafferty et al. (1996)Antibody Engineering, A Practical Approach IRL at Oxford Press, Oxford,England (McCafferty), and Paul (1995) Antibody Engineering ProtocolsHumana Press, Towata, N.J. (Paul). Additional details regarding specificprocedures can be found, e.g., in Ostberg et al. (1983), Hybridoma 2:361-367, Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al., U.S.Pat. No. 4,634,666.

Defining Polypeptides by Immunoreactivity

Because the polypeptides of the invention provide a variety of newpolypeptide sequences (e.g., comprising HA and NA molecules), thepolypeptides also provide new structural features which can berecognized, e.g., in immunological assays. The generation of antiserawhich specifically bind the polypeptides of the invention, as well asthe polypeptides which are bound by such antisera, are features of theinvention.

For example, the invention includes polypeptides (e.g., HA and NAmolecules) that specifically bind to or that are specificallyimmunoreactive with an antibody or antisera generated against animmunogen comprising an amino acid sequence selected from one or more ofthe sequences given herein (e.g., SEQ ID NO: 11-20), etc. To eliminatecross-reactivity with other homologues, the antibody or antisera issubtracted with the HA and/or NA molecules found in public databases atthe time of filing, e.g., the “control” polypeptide(s). Where the othercontrol sequences correspond to a nucleic acid, a polypeptide encoded bythe nucleic acid is generated and used for antibody/antisera subtractionpurposes.

In one typical format, the immunoassay uses a polyclonal antiserum whichwas raised against one or more polypeptide comprising one or more of thesequences corresponding to the sequences herein (e.g., SEQ ID NO:11-20), etc. or a substantial subsequence thereof (i.e., at least about30% of the full length sequence provided). The set of potentialpolypeptide immunogens derived from the present sequences arecollectively referred to below as “the immunogenic polypeptides.” Theresulting antisera is optionally selected to have low cross-reactivityagainst the control hemagglutinin and/or neuraminidase homologues andany such cross-reactivity is removed, e.g., by immunoabsorbtion, withone or more of the control hemagglutinin and neuraminidase homologues,prior to use of the polyclonal antiserum in the immunoassay.

In order to produce antisera for use in an immunoassay, one or more ofthe immunogenic polypeptides is produced and purified as describedherein. For example, recombinant protein can be produced in arecombinant cell. An inbred strain of mice (used in this assay becauseresults are more reproducible due to the virtual genetic identity of themice) is immunized with the immunogenic protein(s) in combination with astandard adjuvant, such as Freund's adjuvant, and a standard mouseimmunization protocol (see, e.g., Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York, for astandard description of antibody generation, immunoassay formats andconditions that can be used to determine specific immunoreactivity).Additional references and discussion of antibodies is also found hereinand can be applied here to defining polypeptides by immunoreactivity.Alternatively, one or more synthetic or recombinant polypeptide derivedfrom the sequences disclosed herein is conjugated to a carrier proteinand used as an immunogen.

Polyclonal sera are collected and titered against the immunogenicpolypeptide in an immunoassay, for example, a solid phase immunoassaywith one or more of the immunogenic proteins immobilized on a solidsupport. Polyclonal antisera with a titer of 10⁶ or greater areselected, pooled and subtracted with the control hemagglutinin and/orneuraminidase polypeptide(s) to produce subtracted pooled titeredpolyclonal antisera.

The subtracted pooled titered polyclonal antisera are tested for crossreactivity against the control homologue(s) in a comparativeimmunoassay. In this comparative assay, discriminatory bindingconditions are determined for the subtracted titered polyclonal antiserawhich result in at least about a 5-10 fold higher signal to noise ratiofor binding of the titered polyclonal antisera to the immunogenicpolypeptides as compared to binding to the control homologues. That is,the stringency of the binding reaction is adjusted by the addition ofnon-specific competitors such as albumin or non-fat dry milk, and/or byadjusting salt conditions, temperature, and/or the like. These bindingconditions are used in subsequent assays for determining whether a testpolypeptide (a polypeptide being compared to the immunogenicpolypeptides and/or the control polypeptides) is specifically bound bythe pooled subtracted polyclonal antisera. In particular, testpolypeptides which show at least a 2-5× higher signal to noise ratiothan the control receptor homologues under discriminatory bindingconditions, and at least about a ½ signal to noise ratio as compared tothe immunogenic polypeptide(s), shares substantial structural similaritywith the immunogenic polypeptide as compared to the known receptor,etc., and is, therefore a polypeptide of the invention.

In another example, immunoassays in the competitive binding format areused for detection of a test polypeptide. For example, as noted,cross-reacting antibodies are removed from the pooled antisera mixtureby immunoabsorbtion with the control polypeptides. The immunogenicpolypeptide(s) are then immobilized to a solid support which is exposedto the subtracted pooled antisera. Test proteins are added to the assayto compete for binding to the pooled subtracted antisera. The ability ofthe test protein(s) to compete for binding to the pooled subtractedantisera as compared to the immobilized protein(s) is compared to theability of the immunogenic polypeptide(s) added to the assay to competefor binding (the immunogenic polypeptides compete effectively with theimmobilized immunogenic polypeptides for binding to the pooledantisera). The percent cross-reactivity for the test proteins iscalculated, using standard calculations.

In a parallel assay, the ability of the control protein(s) to competefor binding to the pooled subtracted antisera is optionally determinedas compared to the ability of the immunogenic polypeptide(s) to competefor binding to the antisera. Again, the percent cross-reactivity for thecontrol polypeptide(s) is calculated, using standard calculations. Wherethe percent cross-reactivity is at least 5-10× as high for the testpolypeptides as compared to the control polypeptide(s) and or where thebinding of the test polypeptides is approximately in the range of thebinding of the immunogenic polypeptides, the test polypeptides are saidto specifically bind the pooled subtracted antisera.

In general, the immunoabsorbed and pooled antisera can be used in acompetitive binding immunoassay as described herein to compare any testpolypeptide to the immunogenic and/or control polypeptide(s). In orderto make this comparison, the immunogenic, test and control polypeptidesare each assayed at a wide range of concentrations and the amount ofeach polypeptide required to inhibit 50% of the binding of thesubtracted antisera to, e.g., an immobilized control, test orimmunogenic protein is determined using standard techniques. If theamount of the test polypeptide required for binding in the competitiveassay is less than twice the amount of the immunogenic polypeptide thatis required, then the test polypeptide is said to specifically bind toan antibody generated to the immunogenic protein, provided the amount isat least about 5-10× as high as for the control polypeptide.

As an additional determination of specificity, the pooled antisera isoptionally fully immunosorbed with the immunogenic polypeptide(s)(rather than the control polypeptide(s)) until little or no binding ofthe resulting immunogenic polypeptide subtracted pooled antisera to theimmunogenic polypeptide(s) used in the immunosorbtion is detectable.This fully immunosorbed antisera is then tested for reactivity with thetest polypeptide. If little or no reactivity is observed (i.e., no morethan 2× the signal to noise ratio observed for binding of the fullyimmunosorbed antisera to the immunogenic polypeptide), then the testpolypeptide is specifically bound by the antisera elicited by theimmunogenic protein.

Nucleic Acid and Polypeptide Sequence Variants

As described herein, the invention provides for nucleic acidpolynucleotide sequences and polypeptide amino acid sequences, e.g.,hemagglutinin and neuraminidase sequences, and, e.g., compositions andmethods comprising said sequences. Examples of said sequences aredisclosed herein (e.g., SEQ ID NO: 1-20). However, one of skill in theart will appreciate that the invention is not necessarily limited tothose sequences disclosed herein and that the present invention alsoprovides many related and unrelated sequences with the functionsdescribed herein, e.g., encoding a HA and/or a NA molecule.

One of skill will also appreciate that many variants of the disclosedsequences are included in the invention. For example, conservativevariations of the disclosed sequences that yield a functionallyidentical sequence are included in the invention. Variants of thenucleic acid polynucleotide sequences, wherein the variants hybridize toat least one disclosed sequence, are considered to be included in theinvention. Unique subsequences of the sequences disclosed herein, asdetermined by, e.g., standard sequence comparison techniques, are alsoincluded in the invention.

Silent Variations

Due to the degeneracy of the genetic code, any of a variety of nucleicacid sequences encoding polypeptides and/or viruses of the invention areoptionally produced, some which can bear lower levels of sequenceidentity to the HA and NA nucleic acid and polypeptide sequences herein.The following provides a typical codon table specifying the geneticcode, found in many biology and biochemistry texts.

TABLE 1 Codon Table Amino acids Codon Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys KAAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser SAGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val VGUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

The codon table shows that many amino acids are encoded by more than onecodon. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU allencode the amino acid arginine. Thus, at every position in the nucleicacids of the invention where an arginine is specified by a codon, thecodon can be altered to any of the corresponding codons described abovewithout altering the encoded polypeptide. It is understood that U in anRNA sequence corresponds to T in a DNA sequence.

Such “silent variations” are one species of “conservatively modifiedvariations,” discussed below. One of skill will recognize that eachcodon in a nucleic acid (except ATG, which is ordinarily the only codonfor methionine, and TTG, which is ordinarily the only codon fortryptophan) can be modified by standard techniques to encode afunctionally identical polypeptide. Accordingly, each silent variationof a nucleic acid which encodes a polypeptide is implicit in anydescribed sequence. The invention, therefore, explicitly provides eachand every possible variation of a nucleic acid sequence encoding apolypeptide of the invention that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code (e.g., as setforth in Table 1, or as is commonly available in the art) as applied tothe nucleic acid sequence encoding a hemagglutinin or a neuraminidasepolypeptide of the invention. All such variations of every nucleic acidherein are specifically provided and described by consideration of thesequence in combination with the genetic code. One of skill is fullyable to make these silent substitutions using the methods herein.

Conservative Variations

Owing to the degeneracy of the genetic code, “silent substitutions”(i.e., substitutions in a nucleic acid sequence which do not result inan alteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence of the invention which encodes an amino acid.Similarly, “conservative amino acid substitutions,” in one or a fewamino acids in an amino acid sequence are substituted with differentamino acids with highly similar properties, are also readily identifiedas being highly similar to a disclosed construct such as those herein.Such conservative variations of each disclosed sequence are a feature ofthe present invention.

“Conservative variations” of a particular nucleic acid sequence refersto those nucleic acids which encode identical or essentially identicalamino acid sequences, or, where the nucleic acid does not encode anamino acid sequence, to essentially identical sequences, see, Table 2below. One of skill will recognize that individual substitutions,deletions or additions which alter, add or delete a single amino acid ora small percentage of amino acids (typically less than 5%, moretypically less than 4%, 3%, 2% or 1%) in an encoded sequence are“conservatively modified variations” where the alterations result in thedeletion of an amino acid, addition of an amino acid, or substitution ofan amino acid with a chemically similar amino acid. Thus, “conservativevariations” of a listed polypeptide sequence of the present inventioninclude substitutions of a small percentage, typically less than 5%,more typically less than 4%, 3%, 2% or 1%, of the amino acids of thepolypeptide sequence, with a conservatively selected amino acid of thesame conservative substitution group. Finally, the addition of sequenceswhich do not alter the encoded activity of a nucleic acid molecule, suchas the addition of a non-functional sequence, is a conservativevariation of the basic nucleic acid.

TABLE 2 Conservative Substitution Groups 1 Alanine (A) Serine (S)Threonine (T) 2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N)Glutamine (Q) 4 Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L)Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan(W)

Unique Polypeptide and Polynucleotide Subsequences

In one aspect, the invention provides a nucleic acid which comprises aunique subsequence in a nucleic acid selected from the sequence of HAand NA molecules disclosed herein, e.g., SEQ ID NO: 1-10. The uniquesubsequence is unique as compared to a nucleic acids corresponding tonucleic acids such as, e.g., those found in GenBank or other similarpublic databases at the time of filing. Alignment can be performedusing, e.g., BLAST set to default parameters. Any unique subsequence isuseful, e.g., as a probe to identify the nucleic acids of the invention.See, above.

Similarly, the invention includes a polypeptide which comprises a uniquesubsequence in a polypeptide selected from the sequence of HA and NAmolecules disclosed herein, e.g., SEQ ID NO: 11-20. Here, the uniquesubsequence is unique as compared to a polypeptide corresponding to,e.g., the amino acid corresponding to polynucleotide sequences found in,e.g., GenBank or other similar public databases at the time of filing.

The invention also provides for target nucleic acids which hybridizeunder stringent conditions to a unique coding oligonucleotide whichencodes a unique subsequence in a polypeptide selected from thesequences of HA and NA molecules of the invention wherein the uniquesubsequence is unique as compared to a polypeptide corresponding to anyof the control polypeptides (sequences of, e.g., the nucleic acidscorresponding to those found in, e.g., GenBank or other similar publicdatabases at the time of filing). Unique sequences are determined asnoted above.

Sequence Comparison, Identity, and Homology

The terms “identical” or percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms described below (or otheralgorithms available to persons of skill) or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides (e.g., DNAs encoding a HA or NA molecule, or theamino acid sequence of a HA or NA molecule) refers to two or moresequences or subsequences that have at least about 90%, preferably 91%,most preferably 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or morenucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using a sequence comparisonalgorithm or by visual inspection. Such “substantially identical”sequences are typically considered to be “homologous,” without referenceto actual ancestry. Preferably, “substantial identity” exists over aregion of the amino acid sequences that is at least about 200 residuesin length, more preferably over a region of at least about 250 residues,and most preferably the sequences are substantially identical over atleast about 300 residues, 350 residues, 400 residues, 425 residues, 450residues, 475 residues, 480 residues, 490 residues, 495 residues, 499residues, 500 residues, 502 residues, 559 residues, 565 residues, or 566residues, or over the full length of the two sequences to be compared.

For sequence comparison and homology determination, typically onesequence acts as a reference sequence to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv Appl Math 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J MolBiol 48:443 (1970), by the search for similarity method of Pearson &Lipman, Proc Natl Acad Sci USA 85:2444 (1988), by computerizedimplementations of algorithms such as GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis., or by visual inspection (see generally,Ausubel et al., supra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J Mol Biol 215:403-410 (1990). Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (www.ncbi.nlm.nih.gov/). Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (see, Altschul etal., supra). These initial neighborhood word hits act as seeds forinitiating searches to find longer HSPs containing them. The word hitsare then extended in both directions along each sequence for as far asthe cumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see, Henikoff & Henikoff (1989) Proc NatlAcad Sci USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc Natl Acad Sci USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Another example of a useful sequence alignment algorithm is PILEUP.PILEUP creates a multiple sequence alignment from a group of relatedsequences using progressive, pairwise alignments. It can also plot atree showing the clustering relationships used to create the alignment.PILEUP uses a simplification of the progressive alignment method of Feng& Doolittle (1987) J. Mol. Evol. 35:351-360. The method used is similarto the method described by Higgins & Sharp (1989) CABIOS 5:151-153. Theprogram can align, e.g., up to 300 sequences of a maximum length of5,000 letters. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster can then be aligned to the next mostrelated sequence or cluster of aligned sequences. Two clusters ofsequences can be aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program can also be used toplot a dendogram or tree representation of clustering relationships. Theprogram is run by designating specific sequences and their amino acid ornucleotide coordinates for regions of sequence comparison.

An additional example of an algorithm that is suitable for multiple DNA,or amino acid, sequence alignments is the CLUSTALW program (Thompson, J.D. et al. (1994) Nucl. Acids. Res. 22: 4673-4680). CLUSTALW performsmultiple pairwise comparisons between groups of sequences and assemblesthem into a multiple alignment based on homology. Gap open and Gapextension penalties can be, e.g., 10 and 0.05 respectively. For aminoacid alignments, the BLOSUM algorithm can be used as a protein weightmatrix. See, e.g., Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci.USA 89: 10915-10919.

Digital Systems

The present invention provides digital systems, e.g., computers,computer readable media and integrated systems comprising characterstrings corresponding to the sequence information herein for the nucleicacids and isolated or recombinant polypeptides herein, including, e.g.,the sequences shown herein, and the various silent substitutions andconservative substitutions thereof. Integrated systems can furtherinclude, e.g., gene synthesis equipment for making genes correspondingto the character strings.

Various methods known in the art can be used to detect homology orsimilarity between different character strings (see, above), or can beused to perform other desirable functions such as to control outputfiles, provide the basis for making presentations of informationincluding the sequences and the like. Examples include BLAST, discussedsupra. Computer systems of the invention can include such programs,e.g., in conjunction with one or more data file or data base comprisinga sequence as noted herein.

Thus, different types of homology and similarity of various stringencyand length between various HA or NA sequences or fragments, etc. can bedetected and recognized in the integrated systems herein. For example,many homology determination methods have been designed for comparativeanalysis of sequences of biopolymers, for spell-checking in wordprocessing, and for data retrieval from various databases. With anunderstanding of double-helix pair-wise complement interactions among 4principal nucleobases in natural polynucleotides, models that simulateannealing of complementary homologous polynucleotide strings can also beused as a foundation of sequence alignment or other operations typicallyperformed on the character strings corresponding to the sequences herein(e.g., word-processing manipulations, construction of figures comprisingsequence or subsequence character strings, output tables, etc.).

Thus, standard desktop applications such as word processing software(e.g., Microsoft Word™ or Corel WordPerfect™) and database software(e.g., spreadsheet software such as Microsoft Excel™, Corel QuattroPro™, or database programs such as Microsoft Access™, Paradox™,GeneWorks™, or MacVector™ or other similar programs) can be adapted tothe present invention by inputting a character string corresponding toone or more polynucleotides and polypeptides of the invention (eithernucleic acids or proteins, or both). For example, a system of theinvention can include the foregoing software having the appropriatecharacter string information, e.g., used in conjunction with a userinterface (e.g., a GUI in a standard operating system such as a Windows,Macintosh or LINUX system) to manipulate strings of characterscorresponding to the sequences herein. As noted, specialized alignmentprograms such as BLAST can also be incorporated into the systems of theinvention for alignment of nucleic acids or proteins (or correspondingcharacter strings).

Systems in the present invention typically include a digital computerwith data sets entered into the software system comprising any of thesequences herein. The computer can be, e.g., a PC (Intel x86 or Pentiumchip-compatible DOS™, OS2™ WINDOWS™ WINDOWSNT™, WINDOWS95™,WINDOWS2000™, WINDOWS98™, LINUX based machine, a MACINTOSH™, Power PC,or a UNIX based (e.g., SUN™ work station) machine) or other commerciallyavailable computer that is known to one of skill. Software for aligningor otherwise manipulating sequences is available, or can easily beconstructed by one of skill using a standard programming language suchas Visualbasic, PERL, Fortran, Basic, Java, or the like.

Any controller or computer optionally includes a monitor which is oftena cathode ray tube (“CRT”) display, a flat panel display (e.g., activematrix liquid crystal display, liquid crystal display), or others.Computer circuitry is often placed in a box which includes numerousintegrated circuit chips, such as a microprocessor, memory, interfacecircuits, and others. The box also optionally includes a hard diskdrive, a floppy disk drive, a high capacity removable drive such as awriteable CD-ROM, and other common peripheral elements. Inputtingdevices such as a keyboard or mouse optionally provide for input from auser and for user selection of sequences to be compared or otherwisemanipulated in the relevant computer system.

The computer typically includes appropriate software for receiving userinstructions, either in the form of user input into a set parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to appropriate language forinstructing the operation, e.g., of appropriate mechanisms or transportcontrollers to carry out the desired operation. The software can alsoinclude output elements for controlling nucleic acid synthesis (e.g.,based upon a sequence or an alignment of sequences herein), comparisonsof samples for differential gene expression, or other operations.

Kits and Reagents

The present invention is optionally provided to a user as a kit. Forexample, a kit of the invention contains one or more nucleic acid,polypeptide, antibody, or cell line described herein (e.g., comprising,or with, a HA and/or NA molecule of the invention). The kit can containa diagnostic nucleic acid or polypeptide, e.g., antibody, probe set,e.g., as a cDNA micro-array packaged in a suitable container, or othernucleic acid such as one or more expression vector. The kit can alsofurther comprise, one or more additional reagents, e.g., substrates,labels, primers, for labeling expression products, tubes and/or otheraccessories, reagents for collecting samples, buffers, hybridizationchambers, cover slips, etc. The kit optionally further comprises aninstruction set or user manual detailing preferred methods of using thekit components for discovery or application of diagnostic sets, etc.

When used according to the instructions, the kit can be used, e.g., forevaluating a disease state or condition, for evaluating effects of apharmaceutical agent or other treatment intervention on progression of adisease state or condition in a cell or organism, or for use as avaccine, etc.

In an additional aspect, the present invention provides system kitsembodying the methods, composition, systems and apparatus herein. Systemkits of the invention optionally comprise one or more of the following:(1) an apparatus, system, system component or apparatus component; (2)instructions for practicing methods described herein, and/or foroperating the apparatus or apparatus components herein and/or for usingthe compositions herein. In a further aspect, the present inventionprovides for the use of any apparatus, apparatus component, compositionor kit herein, for the practice of any method or assay herein, and/orfor the use of any apparatus or kit to practice any assay or methodherein.

Additionally, the kits can include one or more translation system asnoted above (e.g., a cell) with appropriate packaging material,containers for holding the components of the kit, instructionalmaterials for practicing the methods herein and/or the like. Similarly,products of the translation systems (e.g., proteins such as HA and/or NAmolecules) can be provided in kit form, e.g., with containers forholding the components of the kit, instructional materials forpracticing the methods herein and/or the like.

To facilitate use of the methods and compositions of the invention, anyof the vaccine components and/or compositions, e.g., reassorted virus inallantoic fluid, etc., and additional components, such as, buffer,cells, culture medium, useful for packaging and infection of influenzaviruses for experimental or therapeutic vaccine purposes, can bepackaged in the form of a kit. Typically, the kit contains, in additionto the above components, additional materials which can include, e.g.,instructions for performing the methods of the invention, packagingmaterial, and a container.

EXAMPLES Construction and Analysis of H5N1 ca Viruses and Vaccines

Various sequences herein comprising H5N1 HA/NA sequences were used tocreate influenza viruses and vaccines. The HA sequences in such vaccineswere altered from wild-type by removal of the polybasic cleavage sitewithin the HA. The HA/NA sequences were reassorted (in a 6:2reassortment) with A/AA/6/60 (an au, ca virus, see above).

Three strains of H5N1 influenza were used in this example:A/VN/1203/2004, A/HK/491/97, and A/HK/213/2003. Such strains are alsoreferred to within this example as the '97, '03, and '04 strains basedon their year designations. The percent similarity of the HA genes ofsuch three strains is 95-96%. FIG. 1 illustrates modification of thepolybasic cleavage site of an exemplary HA sequence, the '04 HAsequences, used to construct the viruses/vaccines. As stated previously,various embodiments of the invention comprise sequences which havediffering regions of the polybasic cleavage site removed. See above.

As stated, the modified H5N1 sequences (i.e., the modified '97, '03, and'04 genes) were used to construct 6:2 reassortant viruses withA/AA/6/60. It will be appreciated, and is pointed out elsewhere herein,that other desirable backbones could also have been used (e.g., PR8,etc.).

In the 6:2 reassortants of this example, the HA and NA gene sequenceswere derived from the wild type parent virus and the remaining geneswere characterized by sequence analysis as derived from the A/AA/6/60 caparent virus. The reassorted viruses replicated to 8.0-8.5 log₁₀TCID₅₀in eggs. However, it will be appreciated that other embodiments whereinthe log₁₀TCID₅₀ comprises from about 7.0 to about 9.0, from about7.5-8.5, or from about 8.0-8.5 are also claimed within the invention.The cleavability of the modified HA in the constructed viruses byendogenous proteases was restricted in vitro and the viruses weredependent on trypsin (e.g., from about 0.1 ug/ml to about 1.0 ug/ml) forgrowth. The constructed viruses were temperature sensitive in vitro.

The H5N1 ca reassortant viruses (having the modified '97, '03, or '04 HAgenes) were not highly pathogenic for chickens. For example, when4-week-old SPF white Plymouth Rock chickens were inoculatedintravenously with a 1:10 dilution of stock virus (10^(8-8.75)TCID₅₀/ml) and observed for 10 days, it was observed that 8 out of 8chickens died within 1-2 days when wild-type '97, '03, and '04H5N1 wereused, while 0 of 8 chickens died when the H5N1 ca reassortant viruseswere used. As can be seen in FIG. 2, the intranasally administered H5N1ca reassortant viruses did not replicate in chickens.

The H5N1/AA ca reassortants were also not lethal for mice. See FIG. 3,which also shows the TCID₅₀ for the H5N1 wild-type strains. FIG. 4 showsthat the 1997 and 2004H5N1 ca reassortant viruses were restricted inreplication in mice. FIG. 5, shows that the H5N1 ca reassorted virusesare restricted in replication in lungs of mice.

A comparison of the serum HAI antibody titers elicited in mice followinga single intranasal dose of vaccine (2003 ca as compared against 2003wild-type), is shown in FIG. 6.

FIG. 7 shows similar measurements, but using serum neutralizing antibodytiters.

FIG. 8 displays that the H5N1 ca reassortant viruses protect mice fromlethal challenge with 50, 500, or 5,000 LD₅₀ of wild-type H5N1 virus.FIG. 9 shows the efficacy of protection from pulmonary replication ofhomologous and heterologous H5N1 challenge viruses in mice. As can beseen, the ca reassortants replicated less well than the wild-typeviruses did. FIG. 10 shows related data using upper respiratory tractsof mice. Those of skill in the art will be familiar with homologous andheterologous challenges (e.g., testing whether 2003 vaccine protectsagainst a 2003 wild-type challenge (homologous) or whether a 2003vaccine protects against a 1997 wild-type challenge (heterologous),etc.).

FIG. 11 shows efficacy of protection conferred by 2004H5N1 ca vaccineagainst high dose (10⁵TCID₅₀) challenge with homologous or heterologousH5N1 wild-type viruses in mice. FIG. 12 shows efficacy of protectionconferred by 1997 and 2003H5N1 ca vaccines against high dose (10⁵TCID₅₀)challenge with homologous or heterologous H5N1 wild-type viruses inmice. FIG. 13 shows efficacy of protection conferred by 2004H5N1 cavaccine against low or high doses of homologous H5N1 wild-type viruschallenge in mice. FIGS. 11-13 demonstrate that the tested vaccinescould protect against other related viruses.

The current example demonstrates several points concerning exemplaryH5N1 ca reassortant viruses/vaccines of the invention. The modified careassortant '97, '03, and '04 viruses were shown to have in vitro isphenotype, loss of pathogenicity in chickens and attenuation in mice. Itis expected that attenuation is also present in ferrets. Efficacy ofprotection and cross-protection against lethal challenge and systemicspread with wild-type viruses in mice was also shown. Efficacy ofprotection and cross-protections against replication of wild-typechallenge viruses in the respiratory tract of mice is also expected.

It is contemplated to use these (and similar) viruses/vaccines todetermine whether immunogenticy and efficacy is improved following 2doses of vaccine; to assess immunogenicity in non-human primates; toassess attenuation and vaccine efficacy in ferrets; to determine thecontribution of humoral and cellular immunity to observed efficacy ofthe produced vaccines in mice; to determine which residues of the 2003HA contribute to enhanced immunogenicity and introduce them into 1997and 2004 HAs; and to determine the effects of deleting the multibasicamino acid cleavage site and of the gene constellation.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovemay be used in various combinations. All publications, patents, patentapplications, or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application, orother document were individually indicated to be incorporated byreference for all purposes.

1-38. (canceled)
 39. A reassortant influenza virus, wherein said viruscomprises 6 internal genome segments from one or more donor virusesother than A/Ann Arbor/6/60 and a genome segment encoding an immunogenicinfluenza surface antigen comprising the amino acid sequence of SEQ IDNO:
 13. 40. The reassortant influenza virus of claim 39, wherein the 6internal genome segments of the one or more donor viruses are selectedfor comprising one or more phenotypic attributes selected from the groupconsisting of: attenuated, cold adapted and temperature sensitive. 41.The reassortant influenza virus of claim 39, wherein said one or moredonor viruses are PR8.
 42. The reassortant influenza virus of claim 39,wherein said one or more donor viruses are A/Leningrad/17.
 43. Animmunogenic composition comprising an immunologically effective amountof the reassortant influenza virus of claim
 39. 44. An immunogeniccomposition comprising an immunologically effective amount of thereassortant influenza virus of claim
 40. 45. An immunogenic compositioncomprising an immunologically effective amount of the reassortantinfluenza virus of claim
 41. 46. An immunogenic composition comprisingan immunologically effective amount of the reassortant influenza virusof claim
 42. 47. A method for stimulating the immune system of a subjectto produce a protective immune response against influenza virus, themethod comprising administering to the subject an immunologicallyeffective amount of the reassortant influenza virus of claim 39 in aphysiologically effective carrier.
 48. A method for stimulating theimmune system of a subject to produce a protective immune responseagainst influenza virus, the method comprising administering to thesubject an immunologically effective amount of the reassortant influenzavirus of claim 40 in a physiologically effective carrier.
 49. A methodfor stimulating the immune system of a subject to produce a protectiveimmune response against influenza virus, the method comprisingadministering to the subject an immunologically effective amount of thereassortant influenza virus of claim 41 in a physiologically effectivecarrier.
 50. A method for stimulating the immune system of a subject toproduce a protective immune response against influenza virus, the methodcomprising administering to the subject an immunologically effectiveamount of the reassortant influenza virus of claim 42 in aphysiologically effective carrier.
 51. A method of prophylactic ortherapeutic treatment of a viral infection in a subject, the methodcomprising: administering to the subject, the virus of claim 39 in anamount effective to produce an immunogenic response against the viralinfection.
 52. A method of prophylactic or therapeutic treatment of aviral infection in a subject, the method comprising: administering tothe subject, the virus of claim 41 in an amount effective to produce animmunogenic response against the viral infection.
 53. A method ofprophylactic or therapeutic treatment of a viral infection in a subject,the method comprising: administering to the subject, the virus of claim42 in an amount effective to produce an immunogenic response against theviral infection.
 54. The method of claim 51, wherein said virus iskilled or inactivated.
 55. The method of claim 52, wherein said virus iskilled or inactivated.
 56. The method of claim 53, wherein said virus iskilled or inactivated.
 57. The method of claim 47, wherein the subjectis a human.
 58. The method of claim 48, wherein the subject is a human.59. The method of claim 49, wherein the subject is a human.
 60. A liveattenuated influenza vaccine comprising the composition of claim
 43. 61.A live attenuated influenza vaccine comprising the composition of claim44.
 62. A split virus or killed virus vaccine comprising the immunogeniccomposition of claim
 43. 63. A split virus or killed virus vaccinecomprising the immunogenic composition of claim
 44. 64. A method forproducing influenza viruses in cell culture, the method comprising: i)introducing into a population of host cells a plurality of vectorscomprising nucleic acid sequences corresponding to: a) at least 6internal genome segments from a donor virus other than A/Ann Arbor/6/60and at least one genome segment encoding an immunogenic influenzasurface antigen comprising an amino acid sequence of: SEQ ID NO: 13; orb) at least 6 internal genome segments from a donor virus other thanA/Ann Arbor/6/60, wherein said donor virus has one or more phenotypicattributes selected from the group consisting of: attenuated, coldadapted and temperature sensitive; and at least one genome segmentencoding an immunogenic influenza surface antigen comprising an aminoacid sequence of: SEQ ID NO:13; ii) culturing the population of hostcells at a temperature less than or equal to 35° C.; and, iii)recovering a plurality of influenza viruses.
 65. An immunogeniccomposition comprising an immunologically effective amount of theinfluenza virus produced by the method comprising: i) introducing into apopulation of host cells a plurality of vectors comprising nucleic acidsequences corresponding to: a) at least 6 internal genome segments froma donor virus other than A/Ann Arbor/6/60 and a genome segment encodingan immunogenic influenza surface antigen comprising the amino acidsequence of SEQ ID NO: 13; or b) at least 6 internal genome segmentsfrom a donor virus other than A/Ann Arbor/6/60, wherein said donor virushas one or more phenotypic attributes selected from the group consistingof: attenuated, cold adapted and temperature sensitive; and a genomesegment encoding an immunogenic influenza surface antigen comprising theamino acid sequence of SEQ ID NO: 13; ii) culturing the population ofhost cells at a temperature less than or equal to 35° C.; and, iii)recovering a plurality of influenza viruses.
 66. A method forstimulating the immune system of an individual to produce a protectiveimmune response against influenza virus, the method comprisingadministering to the individual an immunologically effective amount ofthe influenza virus produced by the method of claim 64 in aphysiologically effective carrier.
 67. A method for stimulating theimmune system of an individual to produce a protective immune responseagainst influenza virus, the method comprising administering to theindividual the immunogenic composition of claim 65.